Meng Lu, Tang Mingfeng, Zhu Yuxing, Tan Longtao
Research Paper
Knocking-OutTriggers Up-Regulation ofExpression and Enhances Zinc Accumulation in Rice
Meng Lu1, 2, Tang Mingfeng1, 3, Zhu Yuxing1, Tan Longtao1
(410125, China; University of Chinese Academy of Sciences, Beijing 100049, China; Institute of Agricultural Quality Standard and Testing Technology, Chongqing Academy of Agricultural Sciences, Chongqing 401329, China)
Zinc (Zn) is an essential trace mineral that is required for plant growth and development. A number of protein transporters, which are involved in Zn uptake, translocation and distribution, are finely regulated to maintain Zn homeostasis in plant. In this study, we functionally characterized an ATP-binding cassette (ABC) transporter gene,, which is involved in Zn homeostasis.encodes a plasma membrane-localized protein that is expressed mainly in the exodermis and xylem in the rice root.mutants resulted in higher Zn accumulation compared with the wild type. Heterogeneous expression ofin a yeast mutant rescued the Zn-deficiency phenotype, implying transport activity of OsPDR7 to Zn in yeast. However, no ZIP genes except forshowed change in expression profile in themutants, which suggested that OsPDR7 maintains cellular Zn homeostasis through regulatingexpression. RNA-Seq analysis further revealed a set of differentially expressed genes between the wild type andmutants that allowed us to propose a possible OsPDR7-associated signaling network involving transporters, hormone responsive genes, and transcription factors. Our results revealed a novel transporter involved in the regulation of Zn homeostasis and will pave the way toward a better understanding of the fine-tuning of gene expression in the network of transporter genes.
;; zinc; metal accumulation; rice; ATP-binding cassette transporter
Zinc (Zn), a metal ion that serves as a coenzyme, is an essential trace element in all living organisms (Broadley et al, 2007). In plants, Zn is required for diverse biochemical processes including protein synthesis, carbohydrate metabolism, tryptophan and indole-3- acetic acid (IAA) synthesis, membrane integrity, and lipid peroxidation (Broadley et al, 2012). Zn deficiency causes stunted growth and small leaves (Cakmak et al, 1996; Broadley et al, 2007, 2012; Utasee et al, 2022). Conversely, Zn over-accumulation results in toxicity, such as chlorosis in young leaves, and can inhibit photo- synthesis, reduce yield, and stunt growth (Broadley et al, 2007). Therefore, Zn homeostasis is well controlled within a narrow concentration range. A large number of transporters have been identified to be involved in Zn uptake, translocation and re-distribution in plants (Yamaji and Ma, 2014; Swamy et al, 2016; Kawakami and Bhullar, 2018; Tong et al, 2020). These transporters are finely regulated at both the transcriptional and post-transcriptional levels to regulate Zn homeostasis in response to environmental changes and endogenous signals in plants. However, the signaling components and how they are involved in fine tuning Zn homeostasis have not been well studied.
In rice, Zn accumulation requires Zn transporters that belong to ZIP (zinc-regulated transporters, iron- regulated transporter-like protein), HMA (heavy metal ATPase of the P1B-type ATPase) and MTP (metal tolerance protein) families. OsZIP9 is a main Zn influx transporter that functions synergistically withOsZIP5 in Zn uptake in the roots (Huang et al, 2020; Tan et al, 2020; Yang et al, 2020). OsHMA2 (Takahashi et al, 2012; Yamaji et al, 2013), OsZIP4 (Ishimaru et al, 2005, 2007; Mu et al, 2021) and OsZIP7 (Tan et al, 2019) are responsible for the root-to-shoot translocation of Zn, and OsMTP1 (Menguer et al, 2013) and OsHMA3 (Cai et al, 2019) are involved in Zn sequestration into the root vacuoles. OsZIP3, OsZIP7 and OsHMA2play major roles in Zn distribution in the phloem-tropic mode in nodes, and are responsible for the preferential distribution of Zn to the developing tissues (Yamaji and Ma, 2014; Sasaki et al, 2015; Tan et al, 2019). The expression of these transporter genes is finely regulated in response to changes in the environmental Zn status. For example, many OsZIPgenes, such asand, are up-regulated by Zn deficiency and repressed by Zn excess. Knock- out of one of these transporter genes results in changes in the expression of the others (Tan et al, 2019). Recently, two rice F-bZIP transcription factors were reported to complement the zinc deficiency response in, although their exact roles in rice and the regulatory network of ZIP transporters in rice remain to be established (Lilay et al, 2020).
ATP-binding cassette (ABC) transporters are a large family of proteins with canonical trans-membrane domains and nucleotide-binding domains (Higgins and Linton, 2004). They play multiple roles in plant development, organ growth, plant nutrition, and stress responses (Do et al, 2018). Pleiotropic drug resistance (PDR)-type ABC transporters belong to ABC transporter subfamily G and are present only in plants and fungi. There are 15 PDRs inand 22 in rice (Rea, 2007; Verrier et al, 2008; Gupta et al, 2019). PDR proteins are mainly located on the plasma membrane and transport metal ions, secondary metabolites and growth regulators, to regulate plant development and the responses to abiotic and biotic stresses (Gupta et al, 2019). Several transporters from the PDR family are reported to be involved in metal hemostasis control. AtPDR8and AtPDR12 both contribute to Cd or Pb resistance in. AtPDR8 is induced by Cd, Pb and Cu, and pumps them out of the plasma membrane of root epidermal cells in(Kim et al, 2007). Also, AtPDR12 functions as a pump to exclude Pb2+and/or Pb2+-containing toxic compounds from the cytoplasm (Lee et al, 2005). AtPDR8 is also involved in drought and salt resistance and defense against pathogen infection in(Stein et al, 2006; Kim et al, 2010). In rice, two PDR members, OsPDR5 and OsPDR9, are Cd-inducible and confer Cd resistance in yeast, and OsPDR9 has been shown to be an exporter of Cd or Cd conjugates that confers Cd resistance to rice (Oda et al, 2011; Fu et al, 2019).
To identify the genetic components involved in Zn homeostasis control in rice, we performed functional characterization of a set of transporters that are involved in Zn uptake and translocation (Tan et al, 2019, 2020). In this study, we reported the functional characterization of another transporter, OsPDR7, which is involved in the control of Zn homeostasis. We showed that OsPDR7 is a plasma membrane protein that is mainly expressed in the epidermal and xylem regions of the vascular bundle in rice roots. Knock-out ofincreased Zn uptake and consequently the rice plants accumulated significantly more Zn. Heterogeneous expression ofin a yeast mutant rescued the Zn-deficiency phenotype, indicating thatcan transport Zn. However, profiling of OsZIP gene expression and analysis of RNA-Seq data revealed that onlyexpression was up-regulated in therice mutants. These findings suggested that disruption ofdid not hinder Zn uptake and distribution directly, but interfered with the signaling regulation ofexpression may indirectly via.
Expression profile of OsPDR7
The rice PDR family contains 22 members, all of which belong to the ABCG subfamily (Gupta et al, 2019). To investigate their potential roles in micro elemental homeostasis control in rice, we identified 15 PDR mutants from the National Agriculture and Food Research Organization (NARO, https://www.naro.go.jp/ english/index.html) and rice genome-wide knock-out (http://biogle.cn/) libraries that were generated byinsertion or CRISPR/Cas9 knock-out, respectively (Miyao et al, 2003; Lu et al, 2017). Among these, mutations in thegene resulted in Zn over- accumulation in brown rice.is located on the short arm of chromosome 2. Compared with its genomic DNA sequence, the coding sequence ofis 4 326 bp in length, and consist of 23 exons and 22 introns, encoding a protein of 1 441 amino acids. We investigated the expression pattern ofusing various tissues at different stages and seedlings exposed to different metal concentrations with qRT-PCR (Fig. 1-A and -B). The results showed thatis mainly expressed in roots, and that the expression did not change in response to Zn, Fe, Cu, or Mn deficiencies, or Zn excess.
Fig. 1. Expression profile ofand subcellular localization of OsPDR7.
A, Tissue specific gene expression in various tissues at different growth stages. Rice plants were grown under field conditions.was used as the internal standard for gene expression. I, II and III represent the serial numbers of node, leaf blade, leaf sheath and internode from top to bottom of rice plants. Data are Mean ± SD (= 3).
B, Relative expression levelsofin the roots of seedlings exposed to different metal concentrations. The 21-day-old wild type seedlings were transferred to 0.5× Kimura B (KB) solution deficient in Zn, Fe, Cu and Mn (-Zn, -Fe, -Cu and -Mn) or supplemented with 50 μmol/L Zn (Zn-exposed, ++Zn) for 7 d. Data were separately compared with that from seedlings grown in normal 0.5× KB (CK). Values represent Mean ± SD (≥ 3). Statistical comparisons were performed using the Tukey’s HSD mean-separation test at the 0.05 level and no significant difference was observed.
C, Histochemical analysis of transgenic plants expressing thegene driven by thepromoter (a and c) and wild type plants (b and d). (a), Pro::GUS seedling. (b), Wild type seedling. (c), Primary root of Pro::GUS transgenic plant. (d), Primary root of wild type plant. (e–i), Immunostaining with anti-GUS antibody was performed in roots of Pro::GUS transgenic rice seedlings (f–i) and wild type plant (e). Blue indicates autofluorescence emitted from the cell wall due to 4,6-diamidino-2-phenylindole (DAPI) staining. Red indicates the anti-GUS antibody-specific fluorescent signal. (e) Representative transverse sections of (d). (f) and (g), Representative transverse sections of (c). (h) and (i), Representative regions magnified from (g). P, Pericycle; St, Stele; Ep, Exodermis. Scale bars, 1 mm in (a–d), 50 μm in (e-g) and 20 μm in (h) and (i).
D, Subcellular localization of OsPDR7 protein determined in rice protoplasts. For each localization experiment, ≥ 20 individual cells were examined using a Zeiss LSM880 confocal laser scanning microscope (Carl Zeiss, Germany). Scale bars, 2 μm. GFP, Green fluorescent protein.
To investigate the tissue specificity ofexpression in rice, a 2 716 bp DNA fragment encompassing the promoter region ofwas used to drive-() expression in transgenic rice plants. GUS straining analysis showed a strong expression ofin the main roots, lateral roots and coleoptile, but no expression in other tissues (Figs. 1-C and S1). In roots, the GUS activity was very strong in the root tip and gradually became weaker away from the root tip (Fig. 1-C). Cross- sectioning and immunostaining using an anti-GUS antibody and counterstaining with 4,6-diamidino-2- phenylindole (DAPI) for nuclei showed that the GUS signals accumulated in all cells in the root tip, but only in cells of the epidermis, exodermis, and xylem of the vascular bundles in the maturation zone (Fig. 1-C). No signals were detected in the roots of wild type plants, indicating the specificity of the anti-GUS antibody.
We analyzed the subcellular localization of OsPDR7 by using a fusion protein OsPDR7-GFP expressed transiently under the control of the cauliflower mosaic virus (CaMV)promoter in rice protoplasts. The OsPDR7-GFP fluorescence was observed exclusively at the plasma membrane and overlapped with the AtPIP-mCherry signal, which is known to be a plasma membrane marker (Fig. 1-D). This was further confirmed in(Fig. S2). Taken together, these results demonstrated thatencodes a plasma membrane-localized protein that is mainly expressed in rice roots.
Role of OsPDR7 in Zn accumulation in rice
To investigate the physiological role ofin rice, we first characterized three independent knock-out mutant lines (,and) generated using the CRISPR/Cas9 system. DNA sequencing confirmed that the three knock-out lines had different deletions and insertions that caused frame shifts in thecoding sequence (Fig. S3). No obvious vegetative growth aberrance was observed in theknock-out line plants under our conditions but the seed-setting rate and 1000-grain weight ofmutants were reduced compared with those of the wild type (Fig. S4).
When seedlings were cultured in 0.5× Kimura B (KB) solution containing 0.4 μmol/L Zn for 28 d, theknock-out lines accumulated significantly higher levels of Zn in the shoots and roots compared with the wild type (Fig. S5). The Zn distribution ratio between shoots and roots did not change significantly, suggesting that theknock-out lines took up more Zn than the wild type plants. There were no differences in the concentrations of Fe, Mn and Cu in the shoots and roots of theknock-out lines compared with the wild type. We next performed a short-term (2-day) labeling experiment with the stable isotope67Zn (as67ZnSO4) to confirm that the Zn uptake was altered in the knock-out lines. After culture in normal solution for 21 d, seedlings were exposed to 0.4 μmol/L67Zn for 2 d. The67Zn uptake in theknock-out lines increased about 35%–45% in comparison with the wild type (Fig. 2-A). The67Zn concentration in theknock-out lines increased in both the shoots and roots, and the distribution ratio of67Zn was unchanged (Fig. 2-B and -C). As a control, there was no significant change in the uptake and distribution of rubidium (Rb), indicating the normal development of the vascular system (Fig. S6).
Under field conditions, higher Zn levels were detected in the brown rice, rachis, nodeI, node II, leaf sheath I and leaf sheath II in theknock-out lines than in the wild type (Fig. 2-D and -E). We also detected higher Fe levels in the brown rice, node I and leaf sheath I in theknock-out lines under field conditions, possibly as a side effect of the higher Zn accumulation (Fig. S7). The concentrations of Mn and Cu in the brown rice and the other tissues were not significantly different between theknock-out lines and the wild type. To further confirm these observations, we also characterized ainsertion mutant of, which was referred to as. This mutant has atransposon insertion in the 22nd exon (Fig. S8). No obvious differences in agronomic traits were detected betweenmutantsand,and -. The changes in Zn and Fe concentrations inplants were consistent with those in theto -, and the concentrations of Mn and Cu were also not different significantly (Fig. S9).Taken together, these results indicated that knock-out ofenhanced Zn uptake and subsequently Zn accumulation.
We also analyzed the performance ofover- expression plants. The full-lengthcDNA sequence was fused with thepromoter and transformed into Nipponbare genomevia- mediated transformation. Two independent transgenic lines (and) with remarkable increases inexpression were adopted to determine the changes in metal accumulations (Fig. S10). Higher Zn levels were detected in brown rice ofand, however, no significant differences were observed in Zn concentrations of various tissues between thelines and the wild type (Fig. 2-F and -G). Moreover, no obvious differences in Fe, Mn, and Cu concentrations were detected between thelines and the wild type (Fig. S11).
Fig. 2. Zn profiles ofknock-out lines and over- expression lines at vegetative and reproductive growth stages.
A, Total uptake of67Zn by wild type (WT) and threeknock-out lines (to) at the vegetative stage.
B,67Zn concentration in the shoots and roots at the seedling stage.
C,67Zn distribution ratio between the roots and shoots at the seedling stage.
D and E, Zn accumulation in brown rice (D) and in husk, rachis, node, leaf blade and leaf sheath (E) at the reproductive stage.
F and G, Zn accumulation in brown rice (F) and in husk, rachis, node, leaf blade and leaf sheath (G) inover-expression (and) lines at the reproductive stage.
I and II in E and G represent the serial numbers of node, leaf blade and leaf sheath from top to bottom of rice plants.
Seedlings were grown in 0.5× Kimura B (KB) nutrient solution for 21 d, then transferred to 0.5× KB containing 0.4 µmol/L67ZnSO4for 2 d in A–C. Plants were grown in a paddy field until the grains were ripe in D–G. Values represent Mean ± SD (≥ 3). Statistical comparisons against WT were performed with the Tukey’s HSD mean-separation test (*,< 0.05; **,< 0.01).
Zn transport activity in yeast
We carried out a heterologous complementation test in yeast to analyze the Zn transport capability of OsPDR7. The pYES2C- OsPDR7 vector and the empty vector were introduced separately into the Zn uptake-deficient yeast mutant ZHY3 (∆) strain, and the performance of the transgenic yeast strains was tested in solution with a wide range of Zn levels. When glucose was supplied in the medium, there was no difference in cell growth between the yeast ∆strains expressingor the empty vector. When galactose was supplied, the ∆strains expressinggrew better than thoseexpressing the empty vector if the Zn concentration was below 25 μmol/L, but growth was inhibited at higher Zn concentrations (Fig. 3-A). As shown in the yeast growth curve, the Zn levels determined the yeast growth rate, and the yeast cells expressinggrew faster than the control under Zn-deficient conditions, but growth was inhibited in conditions of Zn excess (Fig. 3-B and -C). We also measured the Zn concentration in the yeast cells. The yeast cells expressingaccumulated more Zn than cells harboring the empty vector, but the difference became smaller as the Zn concentration increased (Fig. 3-D). As a control, we also tested the ability of OsPDR7 to transport Cd in yeast strain ∆, which isdefective in a tonoplast transporter and is sensitive to Cd stress (Fig. 3-A). The growth of the ∆yeast cells expressingwas similar to that of the cells carrying the empty vector under Cd treatment, suggesting that OsPDR7 has no Cd transport activity in yeast. Taken together, these results showed that OsPDR7 can transport Zn in yeast.
Expression levels of Zn transporter genes
We compared the expression levels of transporter genes involved in Zn transport, including the OsZIP genes andin theknock-out lines and the wild type to examine how Zn homeostasis is disrupted in theknock-out lines. We found that the expression level ofwas up-regulated by 2- to 3-fold in shoots and roots of themutants (Fig. 4-A), but it is comparable in theover-expression plants and the wild type (Fig. 4-B). In contrast, the expression levels of other Zn transporter genes, such as, did not differ in the roots between theknock-out lines and the wild type (Fig. S12). Meanwhile, the expression levels of Fe-homeostasis related genes, including,,,,and, were also examined. There were no significant changes detected (Fig. S13), confirming a side-effect of Zn increase on Fe accumulation in(Fang et al, 2008; Wang et al, 2020). These results suggested that knock-out ofresulted in up-regulation of, rather than the disruption of Zn homeostasis and the mis-distribution of Zn among rice tissues.
Fig. 3. Complementation assay of OsPDR7 in yeast mutant strain.
A, Zn and Cd transport activity assay of OsPDR7 expressed in yeast. The empty vector pYES2C or OsPDR7 was introduced into Zn uptake-deficient mutant strainor Cd-sensitive mutant strainon the synthetic defined-Ura (SD-U) medium. The yeast strains were cultured on the plate with different Zn or Cd concentrations at 30 for 2–4 d. Zn was chelated with 50 µmol/L ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA).
B and C, Growth rates of yeast cells. Yeast mutant strainexpressingor the empty vector was cultured in SD-U medium containing galactose with 2.5 µmol/L Zn (B) or 500 µmol/L Zn (C).
D, Concentration of Zn in the mutant yeast strainexpressing the empty vector pYES2C or. Data are Mean ± SD (≥ 3).
In B–D, significant differences were determined by one-way analysis of variance (*,< 0.05; **,< 0.01).
Fig. 4.expression in response to exogenous Zn supplied via root culture or foliar spray.
A and B, Expression levels ofin roots and shoots of wild type (WT) and threeknock-out lines (to) (A), as well as in WT andover-expression line () (B). The 28-day-old seedlings were used.
C–E,relative expression levels in local system (C), foliar spray with 0.4 μmol/L67ZnSO4(D), and in control treatments (E) in WT andknockout lines. The 21-day-old seedlings were cultivated in Zn-deficient 0.5× Kimura B (KB) medium for 7 d, followed by 0.4 μmol/L67Zn re-supply to the leaves via foliar spray or 0.5× KB containing 0.4 μmol/L67Zn as the local Zn system for 1 h. The control was 0.01% Trition X-100.
Values represent Mean ± SD (≥ 3). Statistical comparison was performed with the Tukey’s HSD mean-separation test (*,< 0.05; **,< 0.01).
We performed a Zn foliar experiment using hydroponically-grown plants to test whether the up-regulation ofwas dependent on the local Zn status signal in the root or a systemic Zn signal in the shoot (Tan et al, 2020).Seedlings of themutant and the wild type were first grown in Zn-deficient hydroponic solution for 7 d followed by normal hydroponic culture for 21 d. Following this, the plants were supplied with 0.4 μmol/L67ZnSO4in either 0.5× KB solution for root hydroponic incubation or 0.01% Trition X-100 for foliar spray (Fig. 4-C and -D). Under the conditions of Zn deficiency,was highly induced in both theknock-out lines and the wild type, and no differences were detected between them prior to Zn application (Fig. 4-C to -E). The expression ofin roots ofand wild type plants decreased rapidly after Zn resupply both in hydroponic culture or foliar spray containing 0.4 μmol/L67Zn. In hydroponic solution containing 0.4 μmol/L67ZnSO4, the differences inexpression levels between theand wild type plants were detectable within 20 min (Fig. 4-C). For the foliar spray treatment, the differences appeared within 30 min, which was much quicker than 1.5 h that is required for Zn to be transported from the shoot to the root (Fig. 4-D) (Tan et al, 2020). In the control, there was no difference between the expression levels ofin the wild type andmutant when supplied with 0.01% Triton X-100 (Fig. 4-E). These results indicated that knock- out ofdid not impair the local or systematic Zn signaling, and the increase inexpression inplants was independent of these signals.
Transcriptome analysis in ospdr7 mutant and wild type
To further elucidate transcriptomic changes in the osmutant, we conducted RNA-Seq analyses in roots ofand wild typeusing 28-day-old rice seedlings. The change inexpression in thetranscriptome was consistent with the qRT-PCR results in theknock-out lines, indicating the reliability of the transcriptome data. This is further verified by the qRT-PCR analyses of the expressional changes of six randomly selected genes (Fig. S14). We analyzed the differentially expressed genes (DEGs) between the wild type and theknock-out lines and identified 680 up-regulated DEGs and 455 down-regulated DEGs (Fig. 5-A). Next, we performed Gene Ontology (GO) enrichment analysis of each cluster using an FDR (false discovery rate) adjusted≤ 0.05 as the cutoff. This showed that 337 DEGs were highly enriched in several GO terms, including cell wall and cellular component, plasma membrane, nucleotide and nucleic acid metabolic process, binding, molecular function, metabolic process, biological process, response to stress, and transport (Fig. 5-B).
Fig. 5. Differentially expressed genes (DEGs) between wild type (WT) andmutant ().
A, Number of DEGs between WT and.
B, Gene Ontology enrichment analysis of DEGs (≤ 0.05).
C, A heat map showing relative expression of transcription factor, transporter, and hormone responsive-related genes.
JA, Jasmonic acid; IAA, Indole-3-acetic acid; ABA, Abscisic acid.
The roots of WT andseedlings (28-day-old) were collected for transcriptome analysis. To identify DEGs, the expression level of each transcript was calculated using the transcripts per million reads method (= 3).
We found that there were a large number of DEGs that encode transporters, transcription factors, or hormone responsive-related genes, which were divided into three clusters according to their functional annotation (Fig. 5-C). There were six transport-related DEGs between the wild type and.andwere down-regulated, and,,andwere up-regulated in. In addition, there were 12 transcription factor genes that were differentially expressed in theknock-out lines compared to the wild type. Four out of five bZIP transcription factor genes had a positive fold-change in themutant. Three HD-ZIP transcription factor genes were up-regulated in. Four WRKY transcription factor genes were down-regulated in. There were 20 hormone responsive-related DEGs involved in IAA, jasmonic acid (JA), and abscisic acid (ABA) between the wild type and themutant.
At present, a set of transporters that mediate Zn uptake, translocation, and distribution in rice have been identified. The expression levels of these transporter genes were finely regulated by the Zn homeostasis, and changes of the expression of these transporter genes will interfere with Zn homeostasis and trigger changes in the expression of other genes (Muvunyi et al, 2022). However, the genetic network involved in Zn accumulation and homeostasis control has not been fully elucidated. In this study, we functionally characterized a PDR-type ABC transporter gene,, which encodes a plasma-membrane localized protein that is mainly expressed in the roots of rice (Fig. 1). Knock-outchanged the expression level ofand subsequently enhanced Zn uptake and accumulation (Figs. 2 and 4-A).
Members of the ABC transporter family are involved in metal element homeostasis control. ABCC1 and ABCC2 incan bind to Cd and As and sequester them into the vacuole (Park et al, 2012). OsPDR9 and AtPDR8 can pump Cd out of the cell and confer heavy metal resistance to the plant (Kim et al, 2007; Oda et al, 2011; Fu et al, 2019). In this study, we showed thatwas involved in Zn homeostasis control by regulatingexpression. It was revealed that OsPDR7 was an influx transporter for Zn, at least in yeast (Fig. 3). The fact that transgenic plants overexpressingaccumulated more Zn in grains corroborated the transport activity of OsPDR7 to Zn (Fig. 2-F). However, the transport activity was weak because over-expression ofonly resulted in the increase of Zn accumulation in brown rice but not in other tissues (Fig. 2-F and -G). It is puzzling that knock-out of, a root-specially-expressed Zn- influx transporter, resulted in the increase of Zn uptake and accumulation. Interruption of Zn homeostasis in rice plant cells may alter the expression of,,and, in addition to,in a tissue-specific manner (Ishimaru et al, 2005; Chen et al, 2008; Takahashi et al, 2012; Tan et al, 2019, 2020; Mu et al, 2021). In themutant plants, Zn uptake and accumulation increased, while no ZIP transporter genes, with the exception of, showed increased expression in theknock-out lines (Figs. 2-A, 2-B, 4-A and S12). As a result, the increase in Zn accumulation was unlikely due to interruption in Zn distribution among tissues within a plant or among organelles within a cell, but rather to specific up-regulation ofexpression that resulted from knock-out of. It sounded reasonable in the light of the spatially overlapped expression ofandin epidermis, exodermis and vascular bundles in the maturation zones in rice roots (Fig. 2-B) (Huang et al, 2020; Tan et al, 2020; Yang et al, 2020), which was also supported by the observation that the distribution ratio of Zn among tissues did not change, and the up-regulation ofwas not limited to local or systemic signals (Figs. 2-C, 4-C to -E).transcription responds to both local Zn status in the root and systemic signals of Zn status from the shoot (Tan et al, 2020). In this study, we found that changes in the expression ofin theknock-out lines showed the same response to local Zn status from the root and systemic signals of Zn status from the shoot (Fig. 4-C to -E). Taken together, these results suggested that the contribution of the gene itself as a Zn importer to Zn accumulation is trivial and the up-regulation ofexpression contributes mainly to the increases of Zn uptake and accumulation.
The ABCG proteins represent the largest subfamily of the ABC transporter family in rice and. A number of ABCG family members have been reported to be involved in the transport of metal- containing complexes. In this study, we foundOsPDR7 was a Zn transporter, at least in yeast. However, the forms of Zn or Zn-containing substrate for OsPDR7 are still unknown. ABCG transporters play important roles in homeostasis control of hormones, in addition to mineral ions. For example, AtABCG14, AtABCG25, AtABCG37, AtABCG40, OsABCG18 and OsABCG45 function in the transport of cytokinins, ABA and 2,4-D (Ito and Gray, 2006; Kang et al, 2010; Kuromori et al, 2010; Ko et al, 2014; Zhang et al, 2014; Zhao et al, 2019; Zhang et al, 2020). Manipulation of cytokinin dehydrogenase in cereals shows clear impacts on Zn nutrition by regulating the expression ofZIP genes (Gao et al, 2019; Chen et al, 2020). Exogenous ABA applications can decrease the phytotoxic effect of Zn by modulating the transcriptional activity of key genes involved in Zn transport (Song et al, 2019). JA can regulate the expression levels of,andgenes that are involved in Zn, Fe and Cd uptakes (Lei et al, 2020). Therefore, it is tempting to assume that OsPDR7 may transport a Zn-containing compound that may be involved in plant hormone homeostasis control and subsequently regulatesexpression. Indeed, we found several IAA responsive genes in the DEG data (Fig. 5-C). However, Zn is cofactor of IAA synthase (Broadley et al, 2012), and knock-out ofresulted in a Zn increase (Fig. 2). Therefore, investigation into the causal sequence of changes inexpression and IAA responsive genes will help to clarify the signaling network involved in. Alternatively, it would be interesting to find out whether OsPDR7 transports different substrates other than Zn to regulateexpression, since some ABC transporters transport diverse substrates with distinct biochemical features.
At present, litter is known about the signaling components involved in transcriptional regulation of Zn transporter genes, such as. In grapevine (L.), the expression of, an ABA-response regulator, is up-regulated by ABA treatment, and affects the expression of ZIP transporter genes in seedlings (Song et al, 2019). In, bZIP19 and bZIP23 were reported to regulate the expression of Zn-related transporter genes (Assunção et al, 2010). Two rice F-bZIPtranscription factors, OsbZIP48 and OsbZIP50, regulate the Zn deficiency response (Lilay et al, 2020). bZIP transcription factors are a group of ABA-response regulators that can regulate diverse processes including stress signaling (Jakoby et al, 2002). In our transcriptome analysis, there were a number of bZIP, HD-ZIP and WRKY transcription factor genes that were up-regulated in themutant. Elucidation of the signaling pathway in which these transcription factors are involved and identification of their up-stream and down-stream components will facilitate the establishment of the Zn signaling pathway and clarify the mechanism by whichexpression is up-regulated byknock-out.
Rice materials and growth conditions
Three() knock-out lines (,and) were generated using the CRISPR/ Cas9 gene editing system inrice cultivar Nipponbare. A retrotransposoninsertion line in(line no. NG1142), hereafter referred to as, in the Nipponbare genetic background was kindly provided by the NARO (https://www.naro.go.jp/english/index.html). Theover- expression lines were generated via-mediated transformation with the vector of pTCK303-in the genetic background of Nipponbare.
Hydroponic experiments were carried out in 0.5× KB solution (pH 5.8) as described previously (Yamaji et al, 2013; Tan et al, 2020). The seedlings were grown in a floating net in 0.5× KB solution under a 13 h light (28 ºC)/11 h dark (25 ºC) photoperiod for 28 d, and the solution was replaced by every 3 d. In the field experiments, plants were cultured in experimental paddy fields following normal agricultural management at the Institute of Subtropical Agriculture of the Chinese Academy of Sciences in Changsha, Hunan Province, China. Plant tissues such as roots, shoots and grains were harvested separately for measurement of trace mineral element concentration. All experiments were repeated at least three times.
qRT-PCR analyses
RNA was extracted from tissues including the roots, basal stem, leaf blade, leaf sheath at the tilling stage, the roots, basal stem, leaf blade, leaf sheath, immature panicle, spikelet at the booting stage, the roots, basal stem, leaf blade, leaf sheath, node, internode, peduncle, rachis at the flowering stage, the roots, basal stem, leaf blade, leaf sheath, node, internode, peduncle, rachis and husk at the grain filling stage with TRIzol reagent (Sangon Biotech, Shanghai, China), and cDNA was synthesized using the HiScript II First Strand cDNA Synthesis Kit (Vazyme Biotech, Nanjing, China). qRT-PCR analyses were performed by using CHamQTMSYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China). The expression of thegene was used as the internal control for normalization of gene expression. Data were collected using a Roche Light Cycler 480 II real-time PCR instrument (Roche, Switzerland). qRT-PCR primers for genes including, the ZIPgenes andare given in Table S1.
Plasmid construction
Genes and upstream sequences were amplified from Nipponbare mRNA and DNA for vector construction. To generateover-expression plants, the full-lengthcDNA sequence was amplified using the primers 303-OsPDR7-F and 303-OsPDR7-R, and cloned into the PTCK303 vector that was digested withI andI. To construct pEZR(K)-LN-OsPDR7-GFP to determine the subcellular localization of OsPDR7, a cDNA fragment was amplified using the-specific primers OsPDR7- GFP-2F and OsPDR7-GFP-2R, and the DNA fragment was cloned into the pEZR(K)-LN vector that was digested withR I andH I. To construct the yeast expression vector pYES2C-OsPDR7, a cDNA fragment was amplified with the primers YES2C Eco-2F and YES2 Xba-2R, and cloned into the pYES2C vector (Invitrogen, USA) that was digested withR I andI. The promoter region (2 716 bp) of genomic DNA sequence located upstream of theinitiation codon (ATG) was amplified with Phanta Super-Fidelity DNA Polymerase (Vazyme Biotech, Nanjing, China) from Nipponbare genomic DNA using primers OsPDR7-GUS-F and OsPDR7- GUS-R. The promoter-containing sequence was cloned into pCAMBIA1301 using the ClonExpress II One Step Cloning Kit (Vazyme Biotech, Nanjing, China) to construct Pro:: GUS in order to investigate the cell and tissue specificity ofexpression. The sequences of primers are given in Table S1.
Subcellular localization of OsPDR7
A plasma membrane marker pSAT6-mCherry-AtPIP2A and the pEZR(K)-LN-OsPDR7-GFP construct were co-transformed into rice protoplasts. After incubation at 28 ºC in the dark for 12 h, fluorescence was detected with an LSM880 confocal laser scanning microscope (Carl Zeiss, Germany) (Yoo et al, 2007; Tan et al, 2020). The filters were set as follows: EM (emission wavelength) at 488 nm and EX (excitation wavelength) at 535 nm for GFP, and EM at 580nm and EX at 630nm for mCherry. In addition, leaves of one-month-oldseedlings were infiltrated with.strain GV3101 carrying the pEZR(K)-LN-OsPDR7-GFP binary vector and a nuclear marker HY5 vector (Sparkes et al, 2006). After incubation at 28 ºC for 24–36 h, the fluorescence signals were detected as described above.
Yeast metal ion transport activity assay
The pYES2C-OsPDR7 or empty vector was introduced into the Zn uptake-deficient mutant ∆() or Cd-sensitive ∆(BY4741;). The performance of transformed yeast cells was tested in synthetic defined-Ura (SD-Ura) medium containing 2% galactose, 0.67% yeast nitrogen base, 0.2%-Ura supplement, and 2% agar at pH 5.8. For inducible expression, SD-Ura medium containing galactose was supplemented with either 50mmol/L ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA) or 5, 25, or 100mmol/L ZnSO4. After spotting yeast cell dilutions with OD600of 0.2, 0.02, 0.002 and 0.0002 on the media, the plates were incubated for 2–4 d at 30oC, after which the cell growth was recorded using a plant living imaging system FUSION FX7.EDGE SPECTRA (VILBER, France).
To draw the yeast growth curve, the transformed yeast strains were cultured in SD-Ura liquid medium containing glucose. After 1 d of culture, the cells were recovered by centrifugation, washed three times with ddH2O, and the OD600was adjusted to 1.0 using SD-Ura medium containing galactose. The yeast cells were then diluted 100-fold as the initial concentration. The growth of the yeast cells was measured at different times until it reached the plateau stage. All yeast experiments were conducted at least three times independently, and similar results were obtained.
To determine the concentration of Zn in yeast cells, the transformed yeast cells were first cultured in SD-Ura liquid medium containing 2% glucose for 24 h and were enriched by centrifugation and washed three times with ddH2O. The yeast cells were then transferred to SD-Ura liquid medium containing 2% galactose diluted to an initial turbidity value of OD600= 0.1. After the cultures grew to an OD600= 1.0 at 30 ºC, the medium was supplemented with different concentrations of ZnSO4, and the cells were continually cultured in ddH2O for 2 h at 30 ºC. Finally, the cells were rapidly cooled in an ice bath, collected by centrifugation at 4 ºC, and the liquid supernatant was retained. The concentrations of Zn in the supernatant were measured directly with inductively coupled plasma-optical emission spectrometer (ICP-OES-5110, Agilent, USA), and the Zn content reduced from the supernatant was calculated as that absorbed by the yeast cells.
GUS staining assay and immunostaining with anti-GUS antibody
The construct Pro::GUS was transformed into Nipponbare using.-mediated transformation. For GUS staining, tissues including nodes, roots and leaves were incubated in a GUS solution kit (Coolaber, Beijing, China) at 37 ºC overnight. The tissues were then fixed in acetic acid and absolute ethanol (9:1), washed in 95% ethanol five times to remove the chlorophyll, and held in 70% ethanol until observation. Sections of plant tissues were observed using a fluorescence microscope BX51 (Olympus, Japan) following the manufacturer’s instructions.
A rabbit anti-GUS polyclonal antibody (BBI, Shanghai, China) was used as the primary antibody for GUS immunostaining. Roots of wild type and Pro::GUSrice lines were fixed with FAA fixative solution (Coolaber, Beijing, China), followed by paraffin sectioning and immunostaining. The secondary antibody was used at a dilution of 1:500 and the slides were observed using a Zeiss LSM700 confocal laser-scanning microscope (Carl Zeiss, Germany).
Labeling experiment with stable 67Zn isotope
Twenty-one-day-old seedlings of the wild type andknock-out lines were initially grown in 0.5× KB solution and then transferred to 0.5× KB containing 0.4mmol/L67ZnSO4(89.60% enrichment; Isoflex, San Francisco, California, USA) and 1mmol/L RbCl as a control. After 2 d, different leaves, basal node regions and roots were separately sampled for the mineral determination. The samples were washed three times in 5 mmol/L CaCl2and ddH2O before harvest. The67Zn concentration was determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent, USA).
Foliar spray experiments
In the foliar spray experiment, 21-day-old seedlings of the wild type andknock-out lines were cultured in Zn-deficient hydroponic solution for 7 d. The seedlings were then sprayed with a solution containing 0.4mmol/L67ZnSO4and 0.01% Triton X-100 or incubated in a solution containing 0.4mmol/L67ZnSO4. Seedlings sprayed with 0.01% Triton X-100 were used as the control. Total RNA was extracted from roots at time points from 0 to 60 min after foliar spraying.
Determination of metals in plant tissues
All samples harvested were dried at 70 ºC for at least 3 d. The dried samples were digested with 80% HNO3and 20% HClO3at 260 ºC. The mineral elements including Zn, Fe, Mn and Cu were diluted in 1% HNO3and measured using an ICP-OES-5110 instrument (Agilent, USA).
Transcriptome profiling
RNA-Seq profiling was performed using RNA extracted from the roots of 28-day-old seedlings of the wild type andmutant. The expression of each transcript was quantified using Salmon v1.2.1 (Patro et al, 2017). DEGs between the wild type andmutant were identified using the R package DESeq2 v1.32.0 software (Love et al, 2014). GO functional enrichment analysis was carried out on AgriGO (http:// systemsbiology.cau.edu.cn/agriGOv2/index.php). GO enrichment analysis of each cluster using an FDR was adjusted≤ 0.05 as the cutoff. A heat map was constructed for the expression of transcription factors, transporters and hormone responsive- related genes.
ACKNOWLEDGEMENTS
This study was jointly supported by the Key Research and Development Plan of Jiangsu Province, China (Grant No. BE2020318-2) and the National Natural Science Foundation of China (Grant No. U19A2026). We thank the National Agriculture and Food Research Organization (NARO, Japan) for providing the ricemutant line.
SUPPLEMENTAL DATA
The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://www.ricescience.org.
Fig. S1. Tissue expression pattern ofat reproductive stage.
Fig. S2. Subcellular localization of OsPDR7 protein determined in.
Fig. S3. Schematic diagram ofgene structure and CRISPR/Cas9 mutations in three mutant lines.
Fig. S4. Phenotypes of wild type andknock-out line plants at grain filling stage.
Fig. S5.Concentration, uptake and distribution of Zn, Fe, Cu, and Mn in rice shoots and roots at vegetative stage.
Fig. S6. Uptake, concentration and distribution of rubidium (Rb) in shoots and roots at vegetative stage.
Fig. S7. Fe, Cu and Mn concentrations in different tissues inknock-out lines (to) and wild type (WT) under field conditions.
Fig. S8. Genotypic identification and agronomic traits ofinsertion mutant.
Fig. S9. Concentrations of Zn, Fe, Mn and Cu inmutant plants at grain filling stage.
Fig. S10. Expression level ofin roots of over-expression lines (to).
Fig. S11. Fe, Cu and Mn concentrations in different tissues inover-expression lines (and) and wild type (WT) under field conditions.
Fig. S12. Expression levels of OsZIPgenes andin roots ofknock-out lines (to) and wild type (WT).
Fig. S13. Expression levels of Fe-related genes in rootsofknock-out lines (to) and wild type (WT).
Fig. S14. Differentially expressed genes validated by qRT-PCR.
Table S1. List of primers in this study.
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20 May 2022
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http://dx.doi.org/10.1016/j.rsci.2022.05.004
Tan Longtao (tanlongtao@isa.ac.cn)
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