Mol. Cells 2022; 45(5): 294-305
Published online May 31, 2022
https://doi.org/10.14348/molcells.2022.2029
© The Korean Society for Molecular and Cellular Biology
Correspondence to : ihhwang@postech.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
E3 ligase BRUTUS (BTS), a putative iron sensor, is expressed in both root and shoot tissues in seedlings of Arabidopsis thaliana. The role of BTS in root tissues has been well established. However, its role in shoot tissues has been scarcely studied. Comparative transcriptome analysis with shoot and root tissues revealed that BTS is involved in regulating energy metabolism by modulating expression of mitochondrial and chloroplast genes in shoot tissues. Moreover, in shoot tissues of bts-1 plants, levels of ADP and ATP and the ratio of ADP/ATP were greatly increased with a concomitant decrease in levels of soluble sugar and starch. The decreased starch level in bts-1 shoot tissues was restored to the level of shoot tissues of wild-type plants upon vanadate treatment. Through this study, we expand the role of BTS to regulation of energy metabolism in the shoot in addition to its role of iron deficiency response in roots.
Keywords Arabidopsis thaliana, BRUTUS, energy metabolism, shoot tissues
Iron is an essential mineral for all living organisms. Existing in multiple oxidation states, iron acts as a cofactor for many critical enzymes involved diverse biological processes (Kroh and Pilon, 2020; Lee et al., 2012b). On the other hand, excess Fe ions cause the formation of damaging reactive oxygen species (Kaplan and Ward, 2013). Plants therefore have mechanisms to control iron uptake, translocation, assimilation, and bioavailability (Kroh and Pilon, 2019; Hossain et al., 2018; Kobayashi and Nishizawa, 2012). In
In response to iron deficiency, the overall expression of
In contrast to the role of BTS in the root, the role of BTS in maintaining iron homeostasis in the shoot remains elusive. Since iron is a critical cofactor for enzymes in electron transfer and chlorophyll biosynthesis, iron status in the shoot should be important for various reactions, such as photosynthesis in leaf tissues (Kobayashi et al., 2019). A number of studies show that the shoot has a mechanism to sense the level of iron and trigger a phloem-mobile signal that communicates status of iron in the shoot to the root (Grillet et al., 2018; Kobayashi and Nishizawa, 2014; Mendoza-Cózatl et al., 2014). Thus, it is possible that iron status in the shoot plays a role in the control of iron uptake in the root (Enomoto et al., 2007; García et al., 2013; Vert et al., 2003). However, the mechanism concerning iron homeostasis in leaf tissues may be different from that in root tissues. Because of this possible difference in iron homeostasis between leaf and root tissues, the physiological role of BTS in shoot tissues may be different from that in root tissues. In this study, we used transcriptional analysis to investigate BTS function in shoots. Our analysis demonstrated that the lower level of BTS in
The T-DNA insertion line of
The binary construct
To acquire green flourescent protein (GFP) images of the leaves from 5-day-old plants (2 DAT, day after transfer to the iron deficiency condition), a multiphoton microscope (MPM) with a Ti-Sapphire laser (Chameleon Vision II; Coherent, Germary) was used at 140-fs pulse width and 80-MHz pulse repetition rate (TCS SP5 II; Leica). MPM images were acquired and processed by LAS AF Lite (Leica). The filter set had an excitation wavelength/spectral detection bandwidth of 930 nm/500 to 550 nm for GFP.
Total protein extracts were prepared as described previously with some modifications (Lee et al., 2021). Plant leaf tissues were ground in liquid nitrogen and homogenized using extraction buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, EDTA-free protease inhibitor cocktail [Roche, Switzerland]). The mixtures were centrifuged at 18,000 ×
Total RNA was prepared from shoot tissues of Col-0 (wild-type) and
The ‘expressed’ genes were first identified as the ones with FPKM larger than 1 in at least one sample. For those expressed genes, FPKM values were converted to log2-FPKM after adding one to the FPKM values. The log2-FPKMs were then normalized using the quantile normalization method (Bolstad et al., 2003). We then identified DEGs as the ones with absolute log2-fold-changes > 0.58 (1.5-fold) for the comparison of
Total RNA was isolated from leaf tissues using the Qiagen RNeasy mini kit according to the manufacturer’s instructions. RNA was treated with TURBO DNase (Invitrogen). 2 μg of total RNA were converted into single-stranded cDNA using the high-capacity cDNA reverse transcription kit with random hexamer (Applied Biosystems, USA) (Wang et al., 2018). To perform qRT-PCR reactions, PowerUP SYBRGreen Master Mix (Applied Biosystems), cDNA and gene specific primers (Supplementary Table S1) were mixed, and PCR was performed using a cycling protocol of 15 s at 95°C and 60 s at 60°C for 40 cycles.
The level of ADP and ATP, and the ratio of ADP/ATP were measured by the ADP/ATP Ratio Assay Kit (MAK135; Sigma, USA) following the manufacturer’s instruction. Luminescence was detected by the spectrophotometric multi-well plate reader (TECAN, Switzerland). The levels of NADPH, NADP+ and the ratio of NADPH/NADP+ were measured by NADP/NADPH Quantitation Kit (MAK038; Sigma) following the manufacturer’s instruction. Optical density (OD) 450 nm was detected by spectrophotometric multi-well plate reader (TECAN). Total starch contents in shoot tissues were measured by the Starch Colorimetric/Fluorometric Assay Kit (BioVision, USA) following the manufacturer’s instruction. OD 570 nm oxyred signal was detected by the spectrophotometric multi-well plate reader (TECAN). Soluble sugar content was measured by the CheKineTM Plant Soluble Sugar Colorimetric Assay Kit (KTB1320; Abbkine) following the manufacturer’s instruction. OD 620 nm was detected by the spectrophotometric multi-well plate reader (TECAN).
The light induction curves of electron transport rate (ETRII), effective quantum yield of PSII (YII) were measured by a Imaging-PAM chlorophyll fluorometer (Heinz Walz GmbH, Germany) as described previously (Li et al., 2021).
Chlorophyll contents were measured as described previously (Wang et al., 2018). Chlorophyll was extracted using 95% ethanol (v/v) from plant leaves at 4°C overnight in the dark. OD of extracted chlorophyll was measured at 664 nm and 648 nm to determine contents of chlorophyll a and b, respectively. The formula 5.24 × OD664/20 was used to calculate chlorophyll a contents and 22.24 × OD648/20 for chlorophyll b contents. Statistical analysis was performed using Student’s
To elucidate the role of BTS in leaf tissues, we first examined the expression of
To gain insight into the role of BTS in the shoot, we performed RNA sequencing analysis of total RNA from the shoot tissue of Col-0 (wild-type) and
We identified the processes where the
We noted that the expression of a significant number of genes encoded in chloroplast and mitochondrial genomes was increased in
The results of transcriptome analysis showing that the genes involved in ATP synthesis or NADPH synthesis were expressed at higher levels in
Transcriptome analysis showed that photosynthesis-related genes were expressed at higher levels in
Plants accumulate starch in the chloroplast during the day, through photosynthesis in the chloroplast, and transport sucrose, another carbon source, to sink tissues through the phloem (Hennion et al., 2019; Lemoine et al., 2013). Since
While BTS is known to play a critical role in iron deficiency response in root tissue, here we focused on the function of BTS in shoot tissue. In iron homeostasis, iron-related processes may primarily concern the uptake of iron from soil. Thus, it has been suggested that BTS plays a role in preventing the uptake of excess iron from the soil. However, iron-homeostasis-related processes in leaf tissues may primarily concern the distribution of iron that arrives from root tissues. Thus, the role of BTS in iron homeostasis in leaf tissues, if any, may be different from that in root tissues.
A previous study showed that the knock-out mutation of
In shoot tissues, iron plays a crucial role as a cofactor in the form of Fe-S cluster or heme for proteins involved in the electron transport chain reaction of the energy production process and many other processes (Kroh and Pilon, 2020). Thus, one possibility is that BTS is negatively involved in the assimilation of iron into key metabolic enzymes in young leaves. Shoot transcriptome analysis of
More than 80% of the chloroplast gene expression was transcribed by plastid-encoded RNA polymerase (PEP) and the remainder by nucleus-encoded RNA polymerase (PAP) (Zhang et al., 2020). PEP works in a complex with PAPs. Mitochondrial gene expression is regulated through various proteins with DNA-binding or RNA-binding motifs (Lee et al., 2012a; Narsai et al., 2011; Shevtsov et al., 2018). Recently, the mitochondrial transcription termination factor (mTERF) family has been studied for its important function in regulating mitochondrial expression (Shevtsov et al., 2018). However, currently, it is not clearly understood how a lower expression level of BTS leads to up-regulation of these genes encoded by the organellar genomes. Possibly, among many target substrates of BTS, there should be transcription factors or transcription-related factors that control the genes involved in the processes.
Consistent with the higher expression of genes involved in photosynthesis in
In conclusion, we provide evidence that BTS plays an important role in various cellular processes in negative manners. While it negatively regulates iron deficiency responses in the root tissue, it is involved in suppression of genes involved in energy-metabolism-related processes in shoot tissue, suppressing energy metabolism in shoot tissue. In the future, further studies will be necessary to explore the mechanism by which BTS regulates the expression of genes that concern energy-metabolism-related processes in shoot tissue.
This work was supported by the National Research Foundation of Korea (NRF) and funded by a grant from the Korea government (MSIT) (No. 2019R1A2B5B03099982).
B.C. and D.Y.H. performed the experiments and wrote the primary manuscript. I.H. and D.H. designed and supervised the study. T.A.L. and J.L. contributed to the data analysis and revision of the manuscript.
The authors have no potential conflicts of interest to disclose.
Mol. Cells 2022; 45(5): 294-305
Published online May 31, 2022 https://doi.org/10.14348/molcells.2022.2029
Copyright © The Korean Society for Molecular and Cellular Biology.
Bongsoo Choi1 , Do Young Hyeon2
, Juhun Lee1
, Terri A. Long4
, Daehee Hwang2,3
, and Inhwan Hwang1
1Department of Life Science, Pohang University of Science and Technology, Pohang 37673, Korea, 2School of Biological Sciences, Seoul National University, Seoul 08826, Korea, 3Bioinformatics Institute, Seoul National University, Seoul 08826, Korea, 4Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695, USA
Correspondence to:ihhwang@postech.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
E3 ligase BRUTUS (BTS), a putative iron sensor, is expressed in both root and shoot tissues in seedlings of Arabidopsis thaliana. The role of BTS in root tissues has been well established. However, its role in shoot tissues has been scarcely studied. Comparative transcriptome analysis with shoot and root tissues revealed that BTS is involved in regulating energy metabolism by modulating expression of mitochondrial and chloroplast genes in shoot tissues. Moreover, in shoot tissues of bts-1 plants, levels of ADP and ATP and the ratio of ADP/ATP were greatly increased with a concomitant decrease in levels of soluble sugar and starch. The decreased starch level in bts-1 shoot tissues was restored to the level of shoot tissues of wild-type plants upon vanadate treatment. Through this study, we expand the role of BTS to regulation of energy metabolism in the shoot in addition to its role of iron deficiency response in roots.
Keywords: Arabidopsis thaliana, BRUTUS, energy metabolism, shoot tissues
Iron is an essential mineral for all living organisms. Existing in multiple oxidation states, iron acts as a cofactor for many critical enzymes involved diverse biological processes (Kroh and Pilon, 2020; Lee et al., 2012b). On the other hand, excess Fe ions cause the formation of damaging reactive oxygen species (Kaplan and Ward, 2013). Plants therefore have mechanisms to control iron uptake, translocation, assimilation, and bioavailability (Kroh and Pilon, 2019; Hossain et al., 2018; Kobayashi and Nishizawa, 2012). In
In response to iron deficiency, the overall expression of
In contrast to the role of BTS in the root, the role of BTS in maintaining iron homeostasis in the shoot remains elusive. Since iron is a critical cofactor for enzymes in electron transfer and chlorophyll biosynthesis, iron status in the shoot should be important for various reactions, such as photosynthesis in leaf tissues (Kobayashi et al., 2019). A number of studies show that the shoot has a mechanism to sense the level of iron and trigger a phloem-mobile signal that communicates status of iron in the shoot to the root (Grillet et al., 2018; Kobayashi and Nishizawa, 2014; Mendoza-Cózatl et al., 2014). Thus, it is possible that iron status in the shoot plays a role in the control of iron uptake in the root (Enomoto et al., 2007; García et al., 2013; Vert et al., 2003). However, the mechanism concerning iron homeostasis in leaf tissues may be different from that in root tissues. Because of this possible difference in iron homeostasis between leaf and root tissues, the physiological role of BTS in shoot tissues may be different from that in root tissues. In this study, we used transcriptional analysis to investigate BTS function in shoots. Our analysis demonstrated that the lower level of BTS in
The T-DNA insertion line of
The binary construct
To acquire green flourescent protein (GFP) images of the leaves from 5-day-old plants (2 DAT, day after transfer to the iron deficiency condition), a multiphoton microscope (MPM) with a Ti-Sapphire laser (Chameleon Vision II; Coherent, Germary) was used at 140-fs pulse width and 80-MHz pulse repetition rate (TCS SP5 II; Leica). MPM images were acquired and processed by LAS AF Lite (Leica). The filter set had an excitation wavelength/spectral detection bandwidth of 930 nm/500 to 550 nm for GFP.
Total protein extracts were prepared as described previously with some modifications (Lee et al., 2021). Plant leaf tissues were ground in liquid nitrogen and homogenized using extraction buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, EDTA-free protease inhibitor cocktail [Roche, Switzerland]). The mixtures were centrifuged at 18,000 ×
Total RNA was prepared from shoot tissues of Col-0 (wild-type) and
The ‘expressed’ genes were first identified as the ones with FPKM larger than 1 in at least one sample. For those expressed genes, FPKM values were converted to log2-FPKM after adding one to the FPKM values. The log2-FPKMs were then normalized using the quantile normalization method (Bolstad et al., 2003). We then identified DEGs as the ones with absolute log2-fold-changes > 0.58 (1.5-fold) for the comparison of
Total RNA was isolated from leaf tissues using the Qiagen RNeasy mini kit according to the manufacturer’s instructions. RNA was treated with TURBO DNase (Invitrogen). 2 μg of total RNA were converted into single-stranded cDNA using the high-capacity cDNA reverse transcription kit with random hexamer (Applied Biosystems, USA) (Wang et al., 2018). To perform qRT-PCR reactions, PowerUP SYBRGreen Master Mix (Applied Biosystems), cDNA and gene specific primers (Supplementary Table S1) were mixed, and PCR was performed using a cycling protocol of 15 s at 95°C and 60 s at 60°C for 40 cycles.
The level of ADP and ATP, and the ratio of ADP/ATP were measured by the ADP/ATP Ratio Assay Kit (MAK135; Sigma, USA) following the manufacturer’s instruction. Luminescence was detected by the spectrophotometric multi-well plate reader (TECAN, Switzerland). The levels of NADPH, NADP+ and the ratio of NADPH/NADP+ were measured by NADP/NADPH Quantitation Kit (MAK038; Sigma) following the manufacturer’s instruction. Optical density (OD) 450 nm was detected by spectrophotometric multi-well plate reader (TECAN). Total starch contents in shoot tissues were measured by the Starch Colorimetric/Fluorometric Assay Kit (BioVision, USA) following the manufacturer’s instruction. OD 570 nm oxyred signal was detected by the spectrophotometric multi-well plate reader (TECAN). Soluble sugar content was measured by the CheKineTM Plant Soluble Sugar Colorimetric Assay Kit (KTB1320; Abbkine) following the manufacturer’s instruction. OD 620 nm was detected by the spectrophotometric multi-well plate reader (TECAN).
The light induction curves of electron transport rate (ETRII), effective quantum yield of PSII (YII) were measured by a Imaging-PAM chlorophyll fluorometer (Heinz Walz GmbH, Germany) as described previously (Li et al., 2021).
Chlorophyll contents were measured as described previously (Wang et al., 2018). Chlorophyll was extracted using 95% ethanol (v/v) from plant leaves at 4°C overnight in the dark. OD of extracted chlorophyll was measured at 664 nm and 648 nm to determine contents of chlorophyll a and b, respectively. The formula 5.24 × OD664/20 was used to calculate chlorophyll a contents and 22.24 × OD648/20 for chlorophyll b contents. Statistical analysis was performed using Student’s
To elucidate the role of BTS in leaf tissues, we first examined the expression of
To gain insight into the role of BTS in the shoot, we performed RNA sequencing analysis of total RNA from the shoot tissue of Col-0 (wild-type) and
We identified the processes where the
We noted that the expression of a significant number of genes encoded in chloroplast and mitochondrial genomes was increased in
The results of transcriptome analysis showing that the genes involved in ATP synthesis or NADPH synthesis were expressed at higher levels in
Transcriptome analysis showed that photosynthesis-related genes were expressed at higher levels in
Plants accumulate starch in the chloroplast during the day, through photosynthesis in the chloroplast, and transport sucrose, another carbon source, to sink tissues through the phloem (Hennion et al., 2019; Lemoine et al., 2013). Since
While BTS is known to play a critical role in iron deficiency response in root tissue, here we focused on the function of BTS in shoot tissue. In iron homeostasis, iron-related processes may primarily concern the uptake of iron from soil. Thus, it has been suggested that BTS plays a role in preventing the uptake of excess iron from the soil. However, iron-homeostasis-related processes in leaf tissues may primarily concern the distribution of iron that arrives from root tissues. Thus, the role of BTS in iron homeostasis in leaf tissues, if any, may be different from that in root tissues.
A previous study showed that the knock-out mutation of
In shoot tissues, iron plays a crucial role as a cofactor in the form of Fe-S cluster or heme for proteins involved in the electron transport chain reaction of the energy production process and many other processes (Kroh and Pilon, 2020). Thus, one possibility is that BTS is negatively involved in the assimilation of iron into key metabolic enzymes in young leaves. Shoot transcriptome analysis of
More than 80% of the chloroplast gene expression was transcribed by plastid-encoded RNA polymerase (PEP) and the remainder by nucleus-encoded RNA polymerase (PAP) (Zhang et al., 2020). PEP works in a complex with PAPs. Mitochondrial gene expression is regulated through various proteins with DNA-binding or RNA-binding motifs (Lee et al., 2012a; Narsai et al., 2011; Shevtsov et al., 2018). Recently, the mitochondrial transcription termination factor (mTERF) family has been studied for its important function in regulating mitochondrial expression (Shevtsov et al., 2018). However, currently, it is not clearly understood how a lower expression level of BTS leads to up-regulation of these genes encoded by the organellar genomes. Possibly, among many target substrates of BTS, there should be transcription factors or transcription-related factors that control the genes involved in the processes.
Consistent with the higher expression of genes involved in photosynthesis in
In conclusion, we provide evidence that BTS plays an important role in various cellular processes in negative manners. While it negatively regulates iron deficiency responses in the root tissue, it is involved in suppression of genes involved in energy-metabolism-related processes in shoot tissue, suppressing energy metabolism in shoot tissue. In the future, further studies will be necessary to explore the mechanism by which BTS regulates the expression of genes that concern energy-metabolism-related processes in shoot tissue.
This work was supported by the National Research Foundation of Korea (NRF) and funded by a grant from the Korea government (MSIT) (No. 2019R1A2B5B03099982).
B.C. and D.Y.H. performed the experiments and wrote the primary manuscript. I.H. and D.H. designed and supervised the study. T.A.L. and J.L. contributed to the data analysis and revision of the manuscript.
The authors have no potential conflicts of interest to disclose.
Xue Xu, Qiong Zhang, Jiong-yu Hu, Dong-xia Zhang, Xu-pin Jiang, jie-zhi Jia, Jing-ci Zhu, and Yue-sheng Huang