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Article

A Metal Chaperone Gene Regulates Rice Growth and Seed Development by Manganese Acquisition and Homeostasis

by
Chao Li
†,
He Li
†,
Justice Kipkorir Rono
,
Mong Qi Wang
and
Zhi Min Yang
*
Department of Biochemistry and Molecular Biology, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to the work.
Agronomy 2022, 12(7), 1676; https://doi.org/10.3390/agronomy12071676
Submission received: 30 April 2022 / Revised: 12 July 2022 / Accepted: 13 July 2022 / Published: 14 July 2022
(This article belongs to the Section Soil and Plant Nutrition)
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Abstract

:
Manganese (Mn) is a mineral element essential for plant growth and development. In agronomy reality, Mn deficiency or overload in crops disturbs metal homeostasis, photosynthesis, and many other biological processes. Mining genetic resources linking Mn acquisition and homeostasis is vitally important to help understand plant adaptation to Mn stress and breeding genetically improved crops for sustainable agriculture. Metallic chaperone (metallochaperone) is a class of family proteins playing an essential role in positive responses to metal and abiotic stresses. Here, we report a novel function of a metal chaperone gene OsHIPP56 in regulating Mn accumulation in rice (Oryza sativa) crops. OsHIPP56 was transcriptionally induced by excessive Mn stress but hardly by Mn deficiency. OsHIPP56-expression in a yeast Mn-sensitive mutant pmr1 rescued the Mn-defective phenotype by increasing Mn accumulation in cells. Knocking out OsHIPP56 by Crispr/cas9 protocol did not affect the growth and physiological responses of rice seedlings supplied with normal Mn concentration. However, excess Mn stress moderately retarded growth of the knockout plants compared with the wild-type. A life span field trial was conducted under natural conditions with the two rice varieties. Knocking out OsHIPP56 also distorted rice growth, leading to reduced plant height, stem elongation, panicle length, spikelet fertility, seed size, and grain yield. Mn concentrations in rice straw (leaves and stem/internodes), brown rice, and husk in cas9 plants were much lower than those in wild-type. This was particularly seen in the brown rice where the Mn concentrations in cas9 plants were reduced by 26.7–49.1% compared with the wild-type control. Overall, these lines of evidence point out that OsHIPP56 plays a primary role required for rice growth, seed development, and Mn acquisition.

1. Introduction

Manganese (Mn) is a mineral element essential for plant growth and crop productivity owning its active participation in many biological processes such as activation of enzymes, chlorophyll biosynthesis, hormone signaling, and electronic transmission coupled with photosynthesis [1]. In agronomic reality, the bioavailability of Mn ions to crops is not constantly appropriate; and rather, Mn deficiency or poisoning to crops occurs frequently [2]. Thus, maintaining an optimal window of Mn concentrations is critical for healthy crop growth, development, and seed production [3].
Most types of upland soil contain abundant manganese minerals, but the vast majority of insoluble manganese (e.g., inorganic compounds) makes it difficult for crops to absorb it efficiently [4]. Some unfavorable soil conditions such as soil acidity, redox potential, or flooded conditions may drastically increase concentrations of Mn ions up to sufficient or even toxic levels [4]. Rice (Oryza sativa), a gramineous and model plant species, is one of the Mn-tolerant crops because it spends most of its life in flooded or moist soil environments. Many rice genotypes and cultivars are endowed with unique traits contributing to elite Mn homeostasis by translocation and distribution [3]. One of the potent regulators of Mn uptake and allocation in rice is a group of metal transporters, that have been recently identified to ensure an optimal range of Mn concentration and vigorous growth and development [1,3]. OsNramp5 (natural resistance-associated macrophage protein5) functions as a major influx transporter for Mn acquisition from soils to exodermis and endodermis cells [5], while OsMTP9 (metal tolerance protein9) releases Mn from exodermal and endodermal cells as an efflux transporter towards the stele [6]. Both proteins are thought to co-transport of Mn up through xylem. OsYSL2 (yellow stripe 1-like2) is involved in phloem long-distance Mn transport [7], and OsYSL6 mediates Mn tolerance by lowering the concentration of Mn in the apoplastic solution in rice [8]. Additionally, OsMTP8.1 (metal tolerant protein) is involved in Mn detoxification in shoots [9], and OsMTP8.2 mediates Mn tolerance through sequestration of Mn into vacuoles along with OsMTP8.1 [10].
Recent studies show that regulation of metals like Mn and cadmium (Cd) in allocation, detoxification, and homeostasis can be also achieved by some other family proteins [11]. One of them is a special class of metal-binding proteins named metallochaperones that can detoxify cellular toxic metals or chelate non-toxic metals for biological functions [12]. Metallochaperones are intracellular soluble proteins that bind target metal ions and work with some transporters to safely transfer metal ions across cells [13]. Two types of metallochaperones have been categorized, including HPPs (heavy metal-associated plant proteins) and HIPPs (heavy metal-associated isoprenylated plant proteins). The former contains one or two HMA domains and the latter contains an additional C-terminal isoprenylation domain [14,15]. Isoprenylation is a protein modification at cysteine residues in the C-terminal region involved in addition of a hydrophobic anchor for interaction of the protein with membranes or other proteins [16,17]. Most HIPP proteins have been shown to directly regulate heavy metal detoxification and homeostasis [12]. To date, several metallochaperones have been functionally identified in Arabidopsis such as AtATX1, AtCCH, AtCCS, AtHIPP20, AtHIPP21, AtHIPP22, AtHIPP26, and AtHIPP27 [14,16,18,19,20] and rice such as OsHIPP24, OsHIPP29, OsHIPP42, and OsHIPP56 [21,22,23,24]. However, there has been no HIPP member involved in Mn accumulation relevant to rice growth and seed development.
In this study, we functionally characterized a new role of OsHIPP56 in regulation of Mn accumulation and homeostasis in rice. Transcript analysis and yeast assays showed that OsHIPP56 was highly upregulated under Mn stress and accumulated Mn in the cells. Further studies revealed that knocking out OsHIPP56 by Crispr/cas9 protocol compromised rice growth in the early growth period under Mn stress and impaired seed development at the later development stage under the normal Mn supply condition. This study presents evidence that OsHIPP56 is involved in Mn homeostasis under normal Mn-supply conditions, suggesting that OsHIPP56 plays an essential role throughout the rice growth and development.

2. Materials and Methods

2.1. Plant Growth in Hydroponic and Field Experiments

Seeds of rice (Oryza sativa L. Japonica, c.v. Nipponbare) were disinfected with 5% NaClO, washed and germinated for two days. The germinating seedlings were transferred to Kimura B nutrient solution for further growth until two weeks old. The growing rice plants were maintained at 28:25 °C (day: night), a photoperiod of 14:10 h (light: dark) cycle, and 240 mmol m−2 s−1 light intensity. For hydroponic experiments, two-week-old rice plants were grown in the same nutrient solution supplemented with 0.5 (normal control), 250, 500, and 1000 µM Mn for 14 days. Then, plants were photographed, and shoot and root tissues were separately sampled and analyzed with plant height, biomass, chlorophyll content, and metal concentrations.
With regard to field trials, rice seeds were germinated, and seedlings were hydroponically grown in a chamber under the condition indicated above. Four-week-old rice plantlets were transferred and planted in the field paddy soil under natural environmental conditions in Nanjing (yellow brown soil, YBS, 28.23º north latitude and 117.02º east longitude). This study was designed to evaluate seed development and grain yield. Thus, the rice plants were grown until seed ripening and harvested. The trials were designed in completely randomized blocks during the spring and summer (May–September) seasons of 2020–2021. The physical and chemical features of soil in the field were analyzed with the concentrations of major mineral elements including calcium, iron, zinc, manganese, and copper (Table S1). Rice plants were harvested at maturity to obtain different tissues including full plants, leases, stems, brown rice, and husk. A part of tissues was used for Mn measurement and demonstration of agronomic traits such as plant height, length of internodes, tiller number, effective panicle number, panicle length, grain length and width, spikelet fertility, 1000-grain weight, and grain yield.

2.2. mRNA Isolation and Expression Analysis

Rice seedlings were separately collected with root and shoot tissues for RNA extraction using Trizol reagent (Invitrogen). Total RNA was used as a template to synthesize first-strand cDNAs using SuperMix (Transgene) according to the manufacturer’s instructions. Quantitative reverse transcription-quantitative PCR (qRT-PCR) was performed with iTaqTM Universal SYBR Green Supermix (BIO-RAD USA) and gene-specific primers (Table S2), using cycling conditions indicated below. The reaction was run with the following steps: 1 μL cDNA template, 0.8 μL primers, and 10 μL 2 × Hieff qPCR SYBR Green Master Mix (No Rox); 8.2 μL RNase-free Water. The reaction procedure was set to 40 cycles: 94 °C 30 s, 94 °C 5 s, 60 °C 30 s. Experiments were repeated in biological triplicates [25].

2.3. Generation of OsHIPP56 Overexpression and Knockout Rice

The Crispr/cas9 system was used to generate OsHIPP56 mutant lines under the background of Nipponbare. Generation of Crispr/cas9 constructs using the pRGEB31 expression vector was described previously [24]. The resultant Crispr/cas9 constructs were introduced into Agrobacterium tumefaciens strain EHA105 and further transformed into rice embryonic callus to produce transformed rice plantlets according to the previous report [26]. The primers used to identify positive clones are described in Table S2. At least 15 independent T3 generations of Crispr/cas9 lines were obtained. Three lines were randomly selected for functional studies.

2.4. Yeast Complementation Assay

The yeast (Saccharomyces cerevisiae) tolerant function of OsHIPP56 to heavy metal stresses was investigated by expressing the gene in the wild-type strains and its mutants (pmr1, zrc1, and cup2) lacking Mn, Zn, and Cu transport activities [21]. The cloned opening reading frame of OsHIPP56 was constructed into plasmid pYES2 to form OsHIPP56-pYES2 and transformed into the wild-type (BY4741) strain as well as the corresponding mutants pmr1, zrc1, and cup2, which are sensitive to excess Mn, Zn, and Cu stress, respectively. A pYES2 empty vector acted as a control. The transformed yeast cells were cultured overnight at 30 °C in an SD-Ura liquid medium containing 2% Gal and 0.67% YNB. The yeast concentrations were ready for use until 0.6–0.8 in OD600. The overnight suspension was serially diluted with sterile distilled water and spotted (6 μL) onto SD-Ura agar supplemented with Mn (2, 4, or 8 mM), Zn (2, 4, or 8 mM), and Cu (30, 60, or 90 μM) in the presence of Gal. The yeast growth on solid media was maintained at 30 °C for 3 days. In a further experiment to determine metal accumulation, mutants pmr1, zrc1, and cup2 transformed with OsHIPP56-pYES2 or pYES2 empty vector were pre-cultured in SD-Ura liquid medium to OD600 = 0.2–0.4, and then supplied with 2 mM Mn, 200 μM Zn, and 5 μM Cu. Yeast growth was maintained at 30 °C for 2 days, harvested, and determined for metal concentrations.

2.5. Determination of Chlorophyll Content and Electrolyte Leakage

Rice fresh leaves were sampled and immersed in the 80% acetone under darkness for 24 h. Extract of chlorophyll pigments was determined using a spectrophotometer. The 80% acetone was used as a blank control, the absorbance values at 663 and 645 nm were measured, and the total chlorophyll content was calculated. The total chlorophyll content was calculated using a method described previously [27].

2.6. Metal Quantification in Yeast and Plant Tissues

Harvested plant tissues and yeast cells were gently washed in 0.5 mM CaCl2 and dried at 75 °C for 3 d. Dried samples were weighed, digested in nitric acid, diluted in deionized water, and subsequently ion content was determined using ICP-MS (Waltham, MA, USA) according to a previous report [25].

2.7. Statistical Analysis

The data presented here are means of three independent replicates. Statistical analysis was performed on SPSS 22.0. The one-way analysis of variance (ANOVA) and Dunnett’s test were used to compare the mean values (p < 0.05) of controls and treatments, or wild-type and Crispr/cas9 lines.

3. Results

3.1. Expression of OsHIPP56 Was Induced by Excessive Mn Stress

To investigate whether OsHIPP56 was induced by Mn deficiency, two-week-old wild-type rice was deprived of Mn supply and grown for two weeks. Transcript measurement by qRT-PCR showed that OsHIPP56 was slightly expressed in roots and no response was observed in shoots (Figure 1A). Deprivation of Zn and Cu had no effect on OsHIPP56 expression, suggesting that OsHIPP56 may not be involved in Zn and Cu mineral nutrients. We then treated rice seedlings with high Mn concentrations. Unexpectedly, transcripts of OsHIPP56 significantly increased with the concentrations at 0.5 (control), 500, 1000, and 1500 μM Mn for 4 h (Figure 1B). To confirm the stress response, an additional long-term experiment was conducted with rice exposed to 0, 50, 100, 200, and 400 μM Mn for 14 days. A similar result was detected (Figure 1C). These results suggest that OsHIPP56 in rice seedlings can be upregulated by excessive Mn stress.

3.2. Expressing OsHIPP56 in Yeast Conferred Cellular Mn Tolerance and Accumulation

OsHIPP56 cDNAs were expressed in yeast (Saccharomyces cerevisiae) to detect metal tolerance and accumulation under Mn, Zn, and Cu stress. Yeast mutants pmr1, zrc1, and cup2 lacking Mn, Zn, and Cu transport activities and specifically sensitive to the metals, were transformed with pYES2 empty vector and pYES2-OsHIPP56. The transformed and empty (non-transformed) cultures grew similarly in the control medium without metal supplementation. However, the pmr1 cells transformed with OsHIPP56 showed improved growth compared to those carrying the empty vector under Mn stress (Figure 2A). The growth of zrc1 and cup2 cultures transformed with empty vector pYES2 or OsHIPP56 were similar in the presence of high Zn or Cu concentrations, showing that OsHIPP56 did not rescue yeast sensitivity to Zn and Cu (Figure 2B,C). Further studies were performed to investigate OsHIPP56 transport activities by comparing metal accumulations in the transformed and control cultures. The pmr1 cultures harboring OsHIPP56 significantly enhanced Mn accumulation compared to the control (Figure 2D). Similar to growth phenotypes, the Zn and Cu concentrations in zrc1 and cup2 cultures remained unchanged between the control and OsHIPP56-transformants (Figure 2E,F). These results pointed out that OsHIPP56 had an activity of transporting Mn only in the cells.

3.3. Knocking Out OsHIPP56 Compromised Rice Growth at Early Vegetative Stage

The Crispr/cas9 lines were generated to elucidate the physiological role of OsHIPP56 in rice growth under Mn stress at an early stage. Two-week-old rice plants were hydroponically exposed to 0.5 (normal), 250, 500, and 1000 µM Mn for 14 days. No major differences were observed between cas9 and wild-type plants grown in normal Mn (0.5 µM) nutrient solution (Figure 3). When grown under higher Mn stress (particularly at 500 and 1000 µM of Mn), the cas9 plants were more profoundly retarded in growth compared to the wild-type (Figure 3A). Significant reduction in shoot and root growth were detected at 250–1000 and 500–1000 µM Mn, by which the cas9 shoot and root length was reduced by 6.5–13.3% and 6.7–10.0%, respectively, compared to wild-type (Figure 3B,C). Although both shoot and root biomasses were reduced among cas9 and wild-type plants, a marked reduction was only observed in root at 500 µM Mn level (Figure 3D,E).
Chlorophyll is an important trait used to mark the growth response to metal stress [24]. Therefore, leaf chlorophyll concentrations were measured to investigate the response of Crispr/cas9 lines to Mn stress. Both leaf elongation and chlorophyll levels of cas9 and wild-type plants remained unchanged under the basal Mn (0.5 µM) level, but mutation of OsHIPP56 led to marked leaf yellowing and significant reduction in chlorophyll content in cas9 plants (Figure 4A,B). The more severe leaf chlorosis and senescence in cas9 plants were observed following treatment with 250–1000 µM Mn. At 500 μM of Mn, for example, the chlorophyll concentration in cas9 plants was only 50.0–60.0% of the wild-type. These results signified that knocking out OsHIPP56 impaired rice growth and physiological response at the early growth period. The same tissues of rice treated with Mn indicated above were used to measure the Mn concentrations using ICP-MS. Mn concentrations were basically identical in shoots and roots between the cas9 and wild-type plants under normal Mn supply condition; upon exposure to higher Mn levels, the cas9 plants accumulated moderately higher levels of Mn than wild-type (Figure 4C,D).

3.4. Knocking Out OsHIPP56 Impaired Rice Seed Development and Reduced Mn Concentrations in Maturity Rice

To ensure the regulatory role of cas9 plants in rice growth and seed development, both varieties were tested in field soil under natural conditions. Several agronomic traits relevant to seed development and yield compositions were evaluated. Statistical analyses showed that the height of cas9 plants was shortened, with reduction of 8.2–11.8% compared to that of wild-type (Figure 5A,B). Similarly, cas9 plants showed decrement of internode elongation compared with wild-type plants (Figure 5C). The internodes represent the vascular tissues for nutrient delivery and transport; nodes consist of a complex of vascular bundles connecting lower and upper nodes, from which the lower nodes are connected to roots, while the upper nodes are associated with a leaf blade and panicle [28]. The total length of internodes of cas9 plants was reduced by 23.6–31.9% relative to the control (Figure 5D). Further analysis of tiller number and effective panicle number in cas9 plants also showed lower levels than the wild-type (Figure 5E,F). These results suggest that disruption of OsHIPP56 dampened the agronomic traits associated with yield of grains.
The morphological features of seeds and grain composition were further surveyed to reveal variation due to knockout of OsHIPP56. In all characters, significant differences were observed in which three cas9 plants produced smaller sizes of panicle and seed than wild-type (Figure 6A–F). The total grain yield per plants, spikelet fertility, 1000-grain weight, and yield per square meter were significantly decreased in cas9 plants (Figure 6G–K).
Ripening rice tissues and organs were separately sampled, including brown rice, husk, and all other tissues. Concentrations of Mn in the samples were measured. It turned out that the Mn concentrations in cas9 plants were significantly lower those of wild-type (Figure 7). This was particularly seen in the brown rice where the Mn concentrations in cas9 plants were reduced by 26.7–49.1%. These results suggest that knockout of OsHIPP56 impaired Mn accumulation and homeostasis in rice seed.

4. Discussion

Metallochaperones are implicated in diverse functions in plant growth and development, metal recycling and homeostasis, response to pathogen attack, and tolerance to environmental stress [12,15,17,23,26,29]. With regards to metal acquisition, some of metallochaperones respond to one metal or more in plants. For instance, OsHIPP29 detoxified both Zn and Cd [22]. OsATX1 plays an important role in Cd and Cu homeostasis [29]. OsHMP chelates Cd and Mn [26] and OsHIPP33 regulates homeostasis of Zn and Fe [30]. To functionally investigate OsHIPP56, the transcriptional expression of OsHIPP56 under Mn deficiency and excess was first characterized. OsHIPP56 upregulation was strongly induced in the presence of excess Mn compared with the control, but its transcript level was lowered under Mn deficiency. Such a response allowed us to infer that OsHIPP56 would be involved in regulation of rice response to Mn stress. To investigate whether OsHIPP56 has detoxic and metal transport activities, the yeast strains with its mutant (defective Mn tolerance) carrying OsHIPP56 were examined under excessive Mn stress. Heterologous expression in the cells showed that OsHIPP56-transformed yeast conferred higher Mn tolerance and Mn accumulation. This result suggests that OsHIPP56 was able to make yeast cells detoxified under high Mn stress.
To prove the capability of OsHIPP56 function, studies comparing Mn tolerance between knockout and WT plants were undertaken in rice under hydroponic conditions. With the normal or slightly higher Mn supply (0.5 and 250 µM), the growth phenotypes (elongation and biomass weight) remained similar between the cas9 and wild-type plants. However, when Mn concentrations were raised up to higher levels (500 and 1000 µM), cas9 plants had a display of stunted growth including severe loss of chlorophyll and chlorosis symptom in leaf blades. In the meantime, the cas9 plants depicted elevated Mn concentrations in shoots and roots compared with wild-type. This could be the result that disruption of OsHIPP56 failed to chelate excessive Mn ions and to work with some transporters to export Mn out of the cells. This assumption was supported by previous reports indicating the interaction of metallochaperones with transporters sharing similar functions [12]. For instance, ATX1 in Arabidopsis and rice transfers Cu to the plasma membrane where Cu is delivered to the metal transporters AtHMA5, OsHMA4-6, and OsHMA9 for efflux in roots [29,31]. However, unlike metal transporters that sorely mobilize Mn across the plasma membrane or in the cytosol to cellular compartments such as the vacuole, Golgi and endoplasmic reticulum [9,32,33], HIPP is required to bind metals for detoxification first in the cytoplasm and then deliver the metals to transporters to allow metals to cross the plasma membrane [19,25].
The rising population requires modern agriculture to address food security by improving crop yield and seed quality. In this perspective, selection or development of new varieties with desirable plant architecture to increase grain reproductivity is necessitated [34]. The rice architecture is influenced by a series of characteristics such as grains per panicle, panicle morphology, tiller number, tiller angle, and plant height, all of which constitute important agronomic traits for grain potential [35,36]. Several genes governing rice architecture and seed development have been reported. The PROSTRATE GROWTH 1 (PROG1) gene encodes a zinc-finger nuclear transcription factor and controls tiller number and angle [37]. Transgenic rice overexpressing DNA binding with one finger12 (OsDof12) affects plant architecture by reducing rice height, leaf blade, and panicle number, which is associated with hyposensitivity to brassinosteroids [38]. TAC1 (Tiller Angle Controlling) is a primary gene regulating rice tiller angle with potential for application for breeding elite architecture of rice varieties [39]. Additionally, several environmental factors such as nitrogen supply, abiotic stress, and cropping systems also affect rice architecture and seed yield [40,41]. Most genes controlling plant architecture reported are transcription factors associated with hormone signaling pathways [42]. Whether metallochaperones are involved in modification of rice architecture through mineral nutrition and regulators will be the topic of research interest.
This study demonstrated that OsHIPP56 is required for proper rice growth and seed development under natural conditions. Comparative analysis of plant phenotypes revealed that cas9 plants exhibited stunted growth and impaired development, characterized by reduced plant height and stem elongation. Importantly, most agronomic features relevant to grain production such as effective panicle number, panicle length, spikelet fertility, seed size, and grain yield were impaired in the mutant lines. The observation can be explained by long-standing disturbance of Mn uptake and allocation caused by OsHIPP56 mutation, because metal assessment in mature rice showed a general level of Mn in rice straw, brown rice, and husk in cas9 plants. The long-lasting Mn starvation in rice plants most likely weakened the efficiency of photosynthesis and many other important biological processes, as Mn is one of the components in the photosynthetic system II for the redox electron transport chain [1]. These results suggest that Mn concentrations in rice plants must be under control. Currently, Mn uptake in rice roots is mainly governed by OsNRAM5 [5], while translocation of Mn from roots to shoots and distribution at upper nodes to panicles or grains is mediated by some transporters such as OsYSL2 [7]. OsHIPP56 may co-work with the transporters to play that role. Our previous study showed that OsHIPP56 is expressed dominantly in node I, leaf blade, and husk [30], this pattern of OsHIPP56 expression could support the assumption that OsHIPP56 would function with some metal transporters to deliver Mn into seeds. Further investigation on the mechanisms for Mn accumulation in rice grains will be required.

5. Conclusions

This study identified a new function of OsHIPP56 in regulating Mn accumulation in rice. Disruption of OsHIPP56 by the Crispr/cas-9 approach seemingly did not affect the growth of rice seedlings grown in the nutrient solution with a normal supply of Mn. In the long-term field trial, however, disruption of OsHIPP56 was found to impair the growth and seed development. Analysis of agronomic traits revealed the lower grain yield in the cas9 plants with demonstrations of abnormal rice architecture, seed development, and reduced Mn accumulation in grains. Thus, our work pointed out that OsHIPP56 would play an essential role required for regulating rice growth and seed development by maintenance of Mn homeostasis in rice plants.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/agronomy12071676/s1. Table S1. Major texture and physicochemical characteristics of the paddy soils; Table S2. Primer sequences used for this study.

Author Contributions

C.L., H.L. and M.Q.W.: responsible for experiments, methodology, conceptualization, validation, visualization, and data analysis; J.K.R.: modification of manuscript; Z.M.Y.: project administration, resources, supervision, original draft, review, and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Transcriptional expression of OsHIPP56 under the metal stress as analyzed by qRT-PCR. (A) Two-week-old rice plants grew in the normal nutrient solution (control) or nutrient solution without Mn, Zn, and Fe for two weeks. (B) Two-week-old rice plants grew in the nutrient solutions with 0.5 (control), 500, 1000, and 1500 μM Mn for 4 h. (C) Two-week-old rice plants grew in the nutrient solutions with 0.5 (control), 50, 100, 200, and 400 μM Mn for 14 days. Vertical bars represent standard deviation of three biological replicates. Asterisks indicate the significant difference between the control and treatment (* p < 0.05, Dunnett’s test).
Figure 1. Transcriptional expression of OsHIPP56 under the metal stress as analyzed by qRT-PCR. (A) Two-week-old rice plants grew in the normal nutrient solution (control) or nutrient solution without Mn, Zn, and Fe for two weeks. (B) Two-week-old rice plants grew in the nutrient solutions with 0.5 (control), 500, 1000, and 1500 μM Mn for 4 h. (C) Two-week-old rice plants grew in the nutrient solutions with 0.5 (control), 50, 100, 200, and 400 μM Mn for 14 days. Vertical bars represent standard deviation of three biological replicates. Asterisks indicate the significant difference between the control and treatment (* p < 0.05, Dunnett’s test).
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Figure 2. Detoxification of OsHIPP56 expressed in yeast (Saccharomyces cerevisiae) mutants and growth response under the stress of Mn, Zn, and Cu. (A–C) Phenotypes. (D–F) Metal concentrations in mutant cells transformed with empty vector pYES2 or OsHIPP56. The yeast strains BY4741 (wild-type), pmr1, zrc1, and cup2 were transformed with empty vector pYES2 (vector) or OsHIPP56 and incubated overnight in the SD-Ura liquid medium. The cells were diluted by gradient after centrifugation and treated with different concentrations of Mn (control, 2, 4, or 8 mM), Zn (control, 2, 4, or 8 mM), and Cu (control, 30, 60, or 90 μM) in agar media for three days. The metal concentrations in the yeast cells treated with excess Mn (2 mM), Zn (200 μM Zn), and Cu (5 μM) for two days were determined with ICP-MS after sample digestion. Vertical bars represent standard deviation of three biological replicates (20 clones). Asterisks indicate that the mean values are significantly different between the OsHIPP56-transformed cells and empty-vector controls (* p < 0.05, Dunnett’s test).
Figure 2. Detoxification of OsHIPP56 expressed in yeast (Saccharomyces cerevisiae) mutants and growth response under the stress of Mn, Zn, and Cu. (A–C) Phenotypes. (D–F) Metal concentrations in mutant cells transformed with empty vector pYES2 or OsHIPP56. The yeast strains BY4741 (wild-type), pmr1, zrc1, and cup2 were transformed with empty vector pYES2 (vector) or OsHIPP56 and incubated overnight in the SD-Ura liquid medium. The cells were diluted by gradient after centrifugation and treated with different concentrations of Mn (control, 2, 4, or 8 mM), Zn (control, 2, 4, or 8 mM), and Cu (control, 30, 60, or 90 μM) in agar media for three days. The metal concentrations in the yeast cells treated with excess Mn (2 mM), Zn (200 μM Zn), and Cu (5 μM) for two days were determined with ICP-MS after sample digestion. Vertical bars represent standard deviation of three biological replicates (20 clones). Asterisks indicate that the mean values are significantly different between the OsHIPP56-transformed cells and empty-vector controls (* p < 0.05, Dunnett’s test).
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Figure 3. Growth response to Mn stress in the rice wild-type (WT) and Crispr/cas9 (cas9) lines. Two-week-old young rice plants were grown in the nutrient solution supplemented with 0.5, 250, 500, and 1000 µM Mn for 14 days. (A) Growth phenotypes of WT and cas9 lines under Mn stress. (B,C) Shoot height and root length of WT and cas9 lines. (D,E) Dry biomass of shoot and root of WT and cas9 lines. Bars = 3 cm. Vertical bars represent standard deviation of three biological replicates. Asterisks indicate that the mean values are significantly different between the WT and Crispr/cas9 lines (* p < 0.05, Dunnett’s test).
Figure 3. Growth response to Mn stress in the rice wild-type (WT) and Crispr/cas9 (cas9) lines. Two-week-old young rice plants were grown in the nutrient solution supplemented with 0.5, 250, 500, and 1000 µM Mn for 14 days. (A) Growth phenotypes of WT and cas9 lines under Mn stress. (B,C) Shoot height and root length of WT and cas9 lines. (D,E) Dry biomass of shoot and root of WT and cas9 lines. Bars = 3 cm. Vertical bars represent standard deviation of three biological replicates. Asterisks indicate that the mean values are significantly different between the WT and Crispr/cas9 lines (* p < 0.05, Dunnett’s test).
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Figure 4. Analyses of leaf blade growth and chlorophyll responses in wild-type (WT), CRISPR/cas9 (cas9), and overexpression (OE) lines under Mn stress. Two-week-old rice plants were grown in the nutrient solution supplemented with 0.5, 250, 500, and 1000 µM Mn for 14 days. (A,C) Symptoms of Mn toxicity appeared on the fourth-youngest leaf blade of CRISPR/cas9 and OE lines. (B,D) Total chlorophyll concentrations of the third youngest leaf blade. Vertical bars represent standard deviation of three biological replicates. Asterisks indicate that the mean values are significantly different between the WT and cas9 (* p < 0.05, Dunnett’s test).
Figure 4. Analyses of leaf blade growth and chlorophyll responses in wild-type (WT), CRISPR/cas9 (cas9), and overexpression (OE) lines under Mn stress. Two-week-old rice plants were grown in the nutrient solution supplemented with 0.5, 250, 500, and 1000 µM Mn for 14 days. (A,C) Symptoms of Mn toxicity appeared on the fourth-youngest leaf blade of CRISPR/cas9 and OE lines. (B,D) Total chlorophyll concentrations of the third youngest leaf blade. Vertical bars represent standard deviation of three biological replicates. Asterisks indicate that the mean values are significantly different between the WT and cas9 (* p < 0.05, Dunnett’s test).
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Figure 5. Phenotypic analysis of the rice wild-type (WT) and Crispr/cas9 (cas9) lines at the seed maturity period. The rice varieties grew in the field under natural conditions throughout their lifespan. (A) Phenotypes of WT and cas9 lines. (B) Plant height of WT and cas9 lines. (C) Stem elongation of WT and cas9 lines. The interval between two arrows indicates the length of internodes. (D) Length of internode for WT and cas9 lines. (E) Comparison of tiller number per plant between WT and cas9 lines. (F) Effective panicle number per plant comparison between WT and cas9 lines. Bars = 3 cm. Vertical bars represent standard deviation of three biological replicates. Asterisks indicate that the mean values are significantly different between the WT and cas9 lines (* p < 0.05, Dunnett’s test).
Figure 5. Phenotypic analysis of the rice wild-type (WT) and Crispr/cas9 (cas9) lines at the seed maturity period. The rice varieties grew in the field under natural conditions throughout their lifespan. (A) Phenotypes of WT and cas9 lines. (B) Plant height of WT and cas9 lines. (C) Stem elongation of WT and cas9 lines. The interval between two arrows indicates the length of internodes. (D) Length of internode for WT and cas9 lines. (E) Comparison of tiller number per plant between WT and cas9 lines. (F) Effective panicle number per plant comparison between WT and cas9 lines. Bars = 3 cm. Vertical bars represent standard deviation of three biological replicates. Asterisks indicate that the mean values are significantly different between the WT and cas9 lines (* p < 0.05, Dunnett’s test).
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Figure 6. Phenotypic differences in seeds and grain yields for wild-type (WT) and Crispr/cas9 (cas9) lines. The rice varieties were grown in the field under natural conditions. (A–D) Phenotypes of panicle and seed size. (E) Panicle length. (F) Spikelet fertility of a panicle. (G) Yield per plant. (H) The 1000-grain weight. (I) Grain length. (J) Grain width. (K) Seed yield per square meter. Vertical bars represent standard deviation of three biological replicates. Asterisks indicate that the mean values are significantly different between the WT and cas9 lines (* p < 0.05, Dunnett’s test).
Figure 6. Phenotypic differences in seeds and grain yields for wild-type (WT) and Crispr/cas9 (cas9) lines. The rice varieties were grown in the field under natural conditions. (A–D) Phenotypes of panicle and seed size. (E) Panicle length. (F) Spikelet fertility of a panicle. (G) Yield per plant. (H) The 1000-grain weight. (I) Grain length. (J) Grain width. (K) Seed yield per square meter. Vertical bars represent standard deviation of three biological replicates. Asterisks indicate that the mean values are significantly different between the WT and cas9 lines (* p < 0.05, Dunnett’s test).
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Figure 7. Analyses of Mn concentrations in CRISPR/cas9 (cas9), and WT under natural conditions. The rice varieties were grown in the field under natural conditions throughout the lifespan and Mn concentrations in straws, brown rice, and husk were quantified. Vertical bars represent standard deviation of three biological replicates. Asterisks indicate that the mean values are significantly different between the WT and cas9 (* p < 0.05, Dunnett’s test).
Figure 7. Analyses of Mn concentrations in CRISPR/cas9 (cas9), and WT under natural conditions. The rice varieties were grown in the field under natural conditions throughout the lifespan and Mn concentrations in straws, brown rice, and husk were quantified. Vertical bars represent standard deviation of three biological replicates. Asterisks indicate that the mean values are significantly different between the WT and cas9 (* p < 0.05, Dunnett’s test).
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Li, C.; Li, H.; Rono, J.K.; Wang, M.Q.; Yang, Z.M. A Metal Chaperone Gene Regulates Rice Growth and Seed Development by Manganese Acquisition and Homeostasis. Agronomy 2022, 12, 1676. https://doi.org/10.3390/agronomy12071676

AMA Style

Li C, Li H, Rono JK, Wang MQ, Yang ZM. A Metal Chaperone Gene Regulates Rice Growth and Seed Development by Manganese Acquisition and Homeostasis. Agronomy. 2022; 12(7):1676. https://doi.org/10.3390/agronomy12071676

Chicago/Turabian Style

Li, Chao, He Li, Justice Kipkorir Rono, Mong Qi Wang, and Zhi Min Yang. 2022. "A Metal Chaperone Gene Regulates Rice Growth and Seed Development by Manganese Acquisition and Homeostasis" Agronomy 12, no. 7: 1676. https://doi.org/10.3390/agronomy12071676

APA Style

Li, C., Li, H., Rono, J. K., Wang, M. Q., & Yang, Z. M. (2022). A Metal Chaperone Gene Regulates Rice Growth and Seed Development by Manganese Acquisition and Homeostasis. Agronomy, 12(7), 1676. https://doi.org/10.3390/agronomy12071676

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