Research ArticleResearch Article

OsCASP1 Is Required for Casparian Strip Formation at Endodermal Cells of Rice Roots for Selective Uptake of Mineral Elements

Zhigang Wang, Naoki Yamaji, Sheng Huang, Xiang Zhang, Mingxing Shi, Shan Fu, Guangzhe Yang, Jian Feng Ma, Jixing Xia

Published November 2019. DOI: https://doi.org/10.1105/tpc.19.00296

Abstract

In response to diverse environmental conditions, rice (Oryza sativa) roots have developed one Casparian strip (CS) at the exodermis and one CS at the endodermis. Here, we functionally characterized OsCASP1 (Casparian strip domain protein 1) in rice. OsCASP1 was mainly expressed in the root elongation zone, and the protein encoded was first localized to all sides of the plasma membrane of endodermal cells without CS, followed by the middle of the anticlinal side of endodermal cells with CS. Knockout of OsCASP1 resulted in a defect of CS formation at the endodermis and decreased growth under both soil and hydroponic conditions. Mineral analysis showed that the oscasp1 mutants accumulated more Ca, but less Mn, Zn, Fe, Cd, and As in the shoots compared with the wild type. The growth inhibition of the mutants was further aggravated by high Ca in growth medium. The polar localization of the Si transporter Low Si 1 at the distal side of the endodermis was not altered in the mutant, but the protein abundance was decreased, resulting in a substantial reduction in silicon uptake. These results indicated that OsCASP1 is required for CS formation at the endodermis and that the CS in rice plays an important role in root selective uptake of mineral elements, especially Ca and Si.

INTRODUCTION

Concentrations of mineral elements for plant growth and development in soil solution fluctuate spatially and temporally throughout the growth period. For example, the concentration of Mn in paddy soil solution changes more than 100-fold, ranging from submicromolar to hundreds of micromolars depending on soil water conditions (Sasaki et al., 2011). In response to these wide changes of mineral elements in soil, plants have developed a barrier, called the Casparian strip (CS), in the root endodermis, the innermost cortical layer surrounding the central cylinder of roots, that controls the radial movement of mineral elements from soil solution to the stele. The CS consists of a ring-like impregnation of the primary cell wall with lignin (Naseer et al., 2012; Geldner, 2013) and is embedded in the middle of anticlinal cell wall between endodermal cells. It plays an important role in preventing uncontrolled free diffusion of mineral elements from soil solution to the root stele (vascular cylinder) and in blocking leakage of mineral elements from the root back into the soil (Robbins et al., 2014).

Figure1

Recently, great progress has been made in understanding the molecular mechanisms underlying CS formation in Arabidopsis (Arabidopsis thaliana; Lee et al., 2013; Barbosa et al., 2019) and several key genes involved in CS formation have been identified. Formation of CS in Arabidopsis roots is initiated by the localization of CS domain proteins (AtCASPs) at the CS membrane domain, where the CS will be formed (Roppolo et al., 2011). The CS membrane domain anchors the plasma membrane to the cell wall (Alassimone et al., 2010) and recruits the enzymes necessary for lignin biosynthesis, including Peroxidase 64 (PER64), a Respiratory Burst Oxidase Homolog F, and Enhanced Suberin 1 (ESB1), a dirigent-like protein (Hosmani et al., 2013; Lee et al., 2013). CASP1, PER64, and ESB1 are positively regulated by MYB36, a transcription factor (Kamiya et al., 2015; Liberman et al., 2015). On the other hand, the CIF1/2-SGN3-SGN1 signal pathway was proposed to play critical roles in maintaining the integrity of the CS (Doblas et al., 2017; Nakayama et al., 2017). SCHENGEN1 (SGN1), encoding a receptor-like kinase is targeted to the outward-facing surface of endodermal cells (Alassimone et al., 2016), whereas the leucine-rich repeat receptor-like kinase SCHENGEN3 (SGN3) is localized around the CS (Pfister et al., 2014). The peptides CASPARIAN STRIP INTEGRITY FACTOR1 (CIF1) and CIF2 are expressed and sulfated in the stele and thought to diffuse to the endodermis, activating SGN3-dependent closure of the CS (Doblas et al., 2017; Nakayama et al., 2017). Recently, LOTR1 was also reported to be required for CS formation. However, in contrast to CASP1, ESB1, and SGN3, LOTR1 is involved in preventing the ectopic localization of CASP protein to the outside of the normal position of CS deposition (Li et al., 2017). Furthermore, it was reported that a transcription factor, SHR (SHORT-ROOT) acts as a master regulator that regulates almost all aspects of CS formation: position, lignification, and integrity (Li et al., 2018; Barbosa et al., 2019).

The role of the CS in mineral element uptake has been investigated in several mutants defective in CS formation in Arabidopsis. In esb1 and casp1-1 casp3-1 mutants, a significant increase in K, S, Cu, Rb, and Mo and a decrease in Li, Mg, Ca, Mn, Fe, Se, and Sr in the shoots were observed compared with wild type Col-0 when the plants were grown under the same conditions (Hosmani et al., 2013). Knockout of MYB36 caused decreased accumulation of Ca, Mn, and Fe and elevated accumulation of Na, Mg, and Zn in the leaves (Kamiya et al., 2015). The lotr1 mutant exhibited reduced concentrations of Ca, Mn, and Fe and elevated concentrations of K, B, Na, and S in leaves (Li et al., 2017). However, in the sgn3 mutant, most mineral elements except K were largely unaltered (Pfister et al., 2014). The K concentration in the shoots was 1.4- to 3.0-fold lower in the sgn3 mutant than in the wild type. These results indicate that CS exerts a substantial effect on mineral element uptake, although the effect on the ionome is not completely consistent between different studies. These discrepancies in the effect of the CS on the ionome are probably caused by different growth conditions, plant age, and different functions of these genes in CS formation.

Rice (Oryza sativa) is a staple food for half the world’s population. In contrast to Arabidopsis roots, rice roots are characterized by one CS at the exodermis and one CS at the endodermis (Enstone et al., 2002). Furthermore, mature roots have a highly developed aerenchyma, in which almost all of the cortex cells between the exodermis and endodermis are destroyed. Therefore, rice roots have developed an efficient uptake system for radial transport from the soil solution to the stele, which is mediated by influx and efflux transporters localized in both the exodermis and endodermis (Sasaki et al., 2016; Che et al., 2018). In silico simulation modeling revealed that CS plays an important role in efficient silicon uptake of rice (Sakurai et al., 2015). However, little is known about the molecular mechanisms underlying CS formation in rice roots. The precise role of the CS in regulating mineral element uptake in rice is also poorly understood.

In this study, we functionally characterized OsCASP1, a rice homolog of Arabidopsis AtCASP1, in terms of expression pattern, tissue and cellular localization, CS formation, and suberin deposition processes, and performed a phenotypic analysis of mineral element uptake in knockout lines. The rice CS shows a distinct role in controlling the selective uptake of some mineral elements, in contrast to the situation in Arabidopsis.

RESULTS

Sequence Analysis of OsCASP1 and Expression Pattern in Rice

In the rice genome, there are five close uncharacterized homologs of AtCASP1 (Supplemental Figure 1). They share 43 to 48% identity with AtCASP1. RT-qPCR analysis revealed that OsCASP1 showed the highest expression in the roots among five homologs (Figure 1A). This result is consistent with that in a public RNA sequencing database (TENOR, https://tenor.dna.affrc.go.jp; Supplemental Table 1). We generated knockout lines for all OsCASP genes by clustered regularly interspaced short palindromic repeats/ associated protein 9 (CRISPR/Cas9) technology (Supplemental Figure 2) and found that only knockout of OsCASP1 showed a severe growth phenotype (Supplemental Figure 3). Knockout of other OsCASP genes did not affect growth (Supplemental Figure 3). We therefore focused on OsCASP1 in subsequent experiments.

Figure 1.
Figure 1.

Expression Pattern of OsCASP1 in Rice.

(A) Relative expression of five OsCASP genes (OsCASP1, OsCASP2, OsCASP3, OsCASP4, and OsCASP5) in the roots.

(B) Organ-dependent expression of OsCASP1. Different organs from wild-type rice (cv Nipponbare) were sampled at the heading stage and subjected to RNA extraction and expression determination.

(C) Root spatial expression of OsCASP1. RNA was extracted from different segments (0 to 3, 3 to 6, 6 to 9, 9 to 12, and 12 to 15 mm from root apex) of 5-d-old seedlings. Histone H3 was used as an internal standard. Data are means ± sd of three biological replicates.

OsCASP1 (LOC_Os04g58760.1) is composed of three exons and two introns, encodes a protein of 224 amino acids, and belongs to the MARVEL protein family (Supplemental Figure 1A). Based on the SOSUI program (http://bp.nuap.nagoya-u.ac.jp/sosui/), OsCASP1 was predicted to be a membrane protein with four transmembrane domains (Supplemental Figure 1C).

Organ-dependent expression analysis showed that OsCASP1 was highly expressed in the roots (Figure 1B). In the roots, the highest expression was found in the root elongation zone (3 to 6 mm from the apex). In the root tip (0 to 3 mm), OsCASP1 expression was also detected at lower levels, but in the mature root region (>6 mm), expression was very low (Figure 1C).

Spatial Localization of OsCASP1 and CS Formation

To investigate the relationship between OsCASP1 and CS formation, we generated transgenic lines carrying the OsCASP1-GFP fusion gene under the control of the OsCASP1 promoter and performed double staining for lignin as the CS formation marker and GFP. At the root tip region (1 mm from the apex), no GFP signal was observed (Supplemental Figures 4A and 4E). At 3 mm, GFP signal was only observed in the endodermal cells, but no lignin deposition was observed (Figures 2A, 2E, and 2I), indicating that CS formation has not been initiated. Furthermore, OsCASP1 was uniformly distributed at the plasma membrane of endodermal cells at this position. This subcellular localization was confirmed by transient expression of OsCASP1-GFP in rice protoplasts with mCherry-OsRac3, a plasma membrane marker (Chen et al., 2010). The green fluorescence of OsCASP1-GFP was mainly overlapped with the mCherry-OsRac3 signal on the plasma membrane (Supplemental Figure 5).

Figure 2.
Figure 2.

Spatial Localization of OsCASP1 and Casparian Strip Formation.

(A) to (H) Triple staining for GFP (green), cellulose (blue), and lignin (magenta) was performed in the roots of the transgenic plants carrying the ProOsCASP1-OsCASP1-GFP construct. (E), (F), (G), and (H) are magnified 3D images of the yellow box area in (A), (B), (C), and (D), respectively.

(I) to (L) show lignin signal in (E), (F), (G), and (H), respectively. Root cross sections were made at 3 mm (A), 5 mm (B), 15 mm (C), or 30 mm (D) from the apex. Scale bars, 100 μm.

At 5 mm and more from the root apex, lignin deposition at the anticlinal side of the endodermal cells was observed, indicating initiation of CS formation from this region (Figures 2B, 2F, and 2J; Supplemental Figure 4). GFP signal was also only observed at the endodermal cells, but the signal was moved to the middle of anticlinal cell wall between endodermal cells where lignin was deposited (Figures 2B, 2F, and 2J; Supplemental Figure 4). At the mature root region (>10 mm), vascular tissues including xylem and phloem were formed, and GFP signal was also observed at the anticlinal side of the endodermal cells (Figures 2C, 2D, 2G, 2H, 2K, and 2L; Supplemental Figure 4).

To examine whether ProOsCASP1-OsCASP1-GFP is functional in rice, we crossed the oscasp1-1 mutant with the transgenic line carrying the ProOsCASP1-OsCASP1-GFP construct. Among the F2 population, the growth phenotypes of the homozygous oscasp1 carrying the ProOsCASP1-OsCASP1-GFP construct were restored to those of wild-type rice, while those of homozygous oscasp1 lacking the construct were not (Supplemental Figure 6). This result indicates that ProOsCASP1-OsCASP1-GFP is functional.

Involvement of OsCASP1 in CS Formation

To investigate whether OsCASP1 is involved in CS formation, we used two independent knockout lines for OsCASP1 generated by CRISPR/Cas9 technology; one with a 1-bp insertion (oscasp1-1) and the other with a 1-bp deletion (oscasp1-2) at different positions within the first exon (Supplemental Figure 2). We confirmed that the sequences of other OsCASP genes were not changed in the two oscasp1 mutants. CS formation at the exodermis was normal in both the wild type and oscasp1 mutants (Supplemental Figure 7). However, at the endodermis, a defect of CS formation was observed in the mutants. At the mature region of the root (20 mm from the root apex), a restricted, dot-like staining of lignin at the endodermis was observed in the cross section of the wild type (Figures 3A and 3B), while a band-like staining of the anticlinal side of the endodermis was detected in the oscasp1-1 and oscasp1-2 mutants (Figures 3C and 3D). Knockout of OsCASP2, OsCASP3, OsCASP4, or OsCASP5 did not affect the formation of CS (Supplemental Figure 8). These results indicate that only mutation of OsCASP1 affected the correct distribution of lignin deposition on the anticlinal side of the endodermal cell wall.

Figure 3.
Figure 3.

Observation of Casparian Strip Formation in Wild-Type Rice and Knockout Lines of OsCASP1.

(A) to (D) Observation of Casparian strip formation in the wild type (WT) and two mutants. Lignin staining was performed in the roots (20 mm from the apex) of the wild type (A) and (B), oscasp1-1 (C), and oscasp1-2 (D). Magenta shows signal of lignin and blue shows cellulose.

(E) to (M) PI penetration test in the roots of the wild type (E), (F), and (K), oscasp1-1 (G), (H), and (L), and oscasp1-2 (I), (J), and (M) at 10 mm (E) to (J) and 20 mm (K) to (M) from the root apex. Eight-day-old seedlings were exposed to PI in the dark for 1 h. (F), (H), and (J) are magnified images of the yellow boxed area in (E), (G), and (I), respectively. en, Endodermis; x, xylem vessel. Casparian strip is indicated by yellow arrowheads. Scale bars, 20 μm (A) to (D), 100 µm (E), (G), (I), (K), (L), and (M).

Analysis of PI Penetration

To examine whether the function of the endodermal apoplastic barrier was defective in oscasp1, we analyzed the diffusion of propidium iodide (PI), an apoplastic tracer, into the stele. When the roots were exposed to PI for 60 min, in the root tip region (10 mm from the root apex), PI penetrated the apoplastic space of cortex in both the wild type and mutants (Figure 3E, 3G, and 3I), indicating that the CS at the exodermis is not completely formed. However, PI penetration was blocked completely at the endodermis in the wild type, where the CS is located (Figures 3E and 3F). By contrast, penetration of PI into the stele was observed in the mutants (Figures 3G to 3J). These results indicate that apoplastic barrier was disrupted in the mutants. On the other hand, in the mature root region (>20 mm from the root apex), where CS at the exodermis has been formed, PI penetration into the cortex was largely blocked at the exodermis in both the wild-type and mutant roots (Figures 3K to 3M). However, a relatively stronger PI signal was observed in the stele region of the mutants compared with the wild type (Figures 3K to 3M). Since PI taken up by the root tip region will be translocated up to the mature region through xylem, the signal observed in the stele of the mature root region seems to be caused by diffusion of PI derived from the root tip. This is supported by a shorter exposure (40 min) experiment; no PI signal was detected in the stele of the mature root region in either the wild type or the mutants (Supplemental Figure 9), because the PI taken up by the root tip had not reached the mature root region during the shorter time.

Spatial Suberin Deposition in Roots

We examined suberin deposition at different root positions using fluorol yellow 088, a fluorescence suberin dye. At the young root region (15 mm from the root apex), suberin deposition at the exodermis and endodermis was hardly observed in the wild type; however, in two mutants, weak suberin deposition in the endodermis was somewhat observed (Supplemental Figure 10). At root mature region (30 mm), suberin deposition was only patchily observed in both the exodermis and endodermis of the wild type (Supplemental Figure 10). By contrast, strong suberinization of almost the entire endodermis was observed in oscasp1 mutants (Supplemental Figure 10). These data suggest that knockout of OsCASP1 induces earlier and stronger suberin accumulation in the endodermis.

Phenotypic Analysis of oscasp1 Mutants

When grown in hydroponic solution, two mutant lines of OsCASP1 showed retarded growth compared with wild type (Figure 4A). The shoot fresh weight of oscasp1 mutants was ∼60% that of wild type (Figure 4B). The root fresh weight was not significantly decreased (Figure 4C). Growth was also inhibited at the vegetative stage when the plants were grown in different soil under both upland and flooded conditions (Figure 4D; Supplemental Figures 11A to 11D); two mutants showed fewer tillers and necrosis in the older leaves (Figure 4D; Supplemental Figures 11A and 11B). At maturity, the growth and yield of oscasp1 were significantly reduced, including plant height, tiller number, panicle length, and number of grain per plant (Figures 4E to 4I). To confirm that these phenotypes observed in the CRISPR-OsCASP1 lines were caused by disruption of OsCASP1, the oscasp1-1 mutant was complemented with a construct of the OsCASP1 promoter-OsCASP1. The growth phenotype of transgenic plants was recovered to that of wild-type rice (Figure 4E), confirming that the abnormal phenotype observed in the CRISPR-OsCASP1 lines was caused by the mutation in OsCASP1.

Figure 4.
Figure 4.

Phenotypic Analysis of OsCASP1 Knockout Lines.

(A) Phenotype of two independent knockout lines grown in hydroponic solution. Bar, 10 cm.

(B) and (C) Fresh weight of the shoots (B) and roots (C). The wild type, oscasp1-1, and oscasp1-2 were grown in a nutrient solution for 34 d.

(D) Phenotype of two independent knockout lines grown in soil at the vegetative stage. The wild type, oscasp1-1, and oscasp1-2 were grown under flooded condition for 1 month. Bar, 10 cm.

(E) Complementation test. The wild type, oscasp1-1, and two independent complementation lines carrying an OsCASP1 genomic fragment were grown until ripening under flooded condition. Bar, 10 cm.

(F) to (I) Comparison of agronomic traits of wild-type (WT) and oscasp1 plants. Tiller number per plant (F), plant height (G), panicle length (H), and grain number per plant (I). Bar, 5 cm. The data are presented as means ± sd (n = 3). Significant difference was determined by Tukey’s test and labeled with different letters (P < 0.05).

Comparison of the Mineral Element Profile between the Wild Type and oscasp1 Mutant

We compared the mineral element profiles in the shoots and roots between the wild type and two independent oscasp1 mutants grown in hydroponic solution. Before harvest, the plants were exposed to a solution containing Ge, Sr, and Rb, which are chemical analogs of Si, Ca and K, respectively, as well as As and Cd for 1 d. The concentration of Ca and Sr in the shoots was significantly higher in the mutants than in the wild type, and the concentration of P in the shoots was slightly higher in the mutants than in the wild type (Figure 5A). A similar trend was observed in the plants grown under upland and flooded soil conditions (Supplemental Figures 11D and 11E). However, the concentration of As, Cd, Fe, Ge, Mn, and Zn in the shoots was lower in the mutants than in wild-type rice (Figure 5B). The concentration of Mg, K, Rb, and Cu in the shoots was hardly altered in the mutant lines under the conditions tested (Figure 5C). Surprisingly, the concentration of mineral elements except Zn in the roots was similar between the wild type and mutants (Supplemental Figure 12). The mutants showed a higher Zn concentration in the roots compared with the wild type.

Figure 5.
Figure 5.

Mineral Elemental Concentration in the Shoots of Wild-Type Rice and Two OsCASP1 Knockout Lines.

(A) Concentration of Ca, Sr, and P in the shoots.

(B) Concentration of As, Cd, Fe, Ge, Mn, and Zn in the shoots.

(C) Concentration of K, Mg, Pb, and Cu in the shoots. Both wild-type rice (WT) and the knockout lines were cultivated in a nutrient solution for 33 d and then transferred to the same solution in the presence of Ge, Sr, Rb, As, and Cd for 1 d before harvest. The data are presented as means ± sd (n = 3). Significant difference was determined by Tukey’s test and labeled with different letters (P < 0.05).

We also compared the ionome between five oscasp mutants and their wild types. In contrast to the oscasp1 mutants, the difference in mineral element concentrations between oscasp2, oscasp3, oscasp4, and oscasp5 mutants and their wild types was small in both the roots and shoots (Supplemental Figure 13).

Si Uptake and Polarity of the Si Transporter Lsi1 in oscasp1 Mutants

Rice is a typical Si-accumulating plant. The above analysis showed that the concentration of Ge, a chemical analog of Si, was decreased by about half in the mutants during a 1-d exposure (Figure 5B). We therefore performed a time-course experiment on Si uptake in the mutants and the wild type. Whereas water uptake did not show a large difference between the wild type and two mutants (Figure 6A), Si uptake was significantly lower in the mutants than in the wild type at each time point (Figure 6B).

Figure 6.
Figure 6.

Effect of Knockout of OsCASP1 on Silicon Uptake, and Expression and Localization of the Silicon Transporter Lsi1 in Rice.

(A) and (B) Time-dependent uptake of water (A) and Si (B).

(C) Expression of Lsi1 in roots of both the wild type (WT) and oscasp1 mutants. RNA was extracted from different segments of the roots (0 to 6, 6 to 12, and 12 to 18 mm from root apex) of 5-d-old seedlings of the wild type and oscasp1 mutants. Histone H3 was used as an internal standard. Data are means ± sd of three biological replicates. Significant difference was determined by Tukey’s test and labeled with different letters (P < 0.05).

(D) to (G) Localization of Lsi1 in the wild type and oscasp1 mutants. Triple staining for Lsi1 (green), cellulose (blue), and lignin (red) was performed in the seminal roots of the wild type (D) and (E), oscasp1-1 (F), and oscasp1-2 (G). (E) Magnified image of the yellow boxed area in (D). Root cross sections were made at 20 mm from the apex. Scale bars, 100 μm.

Si uptake is mediated by an influx transporter Lsi1 and an efflux transporter Lsi2, which are polarly localized at the distal and proximal side of both the exodermis and endodermis (Ma et al., 2006, 2007). Knockout of OsCASP1 did not affect Lsi1 expression in the different segments of the roots (Figure 6C). To examine the effect of OsCASP1 knockout on Lsi1 polarity, we performed triple staining of the roots for Lsi1, lignin, and cellulose in both the wild type and mutants. The polarity of Lsi1 was not altered in the oscasp1 mutants; the transporter was polarly localized at the distal side of the endodermis in both the wild type and oscasp1 mutants (Figures 6D to 6G). However, the Lsi1 abundance was significantly reduced in the mutants (Figures 6D to 6G).

Effect of Different Ca Concentrations on Ca Accumulation and Growth of oscasp1 Mutants

Although knockout of OsCASP1 altered the mineral element accumulation in the shoots (Figure 5), the most striking change was of Ca accumulation in the shoot of the mutants. To test whether high Ca accumulation is a major factor causing growth retardation, we grew the wild type and two independent mutants at different Ca levels. Growth of the wild type was hardly affected by an increasing Ca supply from 0.18 mm to 10 mm (Figures 7A to 7F; Supplemental Figure 14). However, the growth of mutants was inhibited more with an increasing Ca supply (Figures 7A to 7F; Supplemental Figure 14). The Ca concentration in the mutant shoots was 1.5, 2.1, 2.2, and 3.4 times that of the wild type, respectively, at 0.18, 1, 5, and 10 mM Ca (Figure 7G). However, there was no difference in the root Ca concentration between the wild type and two oscasp1mutants (Figure 7H). The Ca concentration in the xylem sap of oscasp1 significantly increased, with increasing external Ca concentrations, while those in the wild-type rice nearly saturated (Figure 7I). These results indicate that CS plays an important role in controlling Ca movement to the stele and that the growth inhibition in the mutants was largely caused by over accumulation of Ca in the shoots.

Figure 7.
Figure 7.

Growth of OsCASP1 Knockout Lines at Different Ca Concentrations.

(A) to (D) Growth of wild-type rice (WT) and two OsCASP1 knockout lines. Bar, 10 cm.

(E) and (F) Dry weight of the shoots and roots.

(G) and (H) Ca concentration in the shoots (G) and roots (H). Seedlings (2 weeks old) were grown in a nutrient solution containing 0.18, 1, 5, or 10 mm CaCl2 for 12 d.

(I) Ca concentration in the xylem sap. Xylem sap was collected from the wild type and knockout lines exposed to different Ca concentrations (0.18, 1, 5, and 10 mm) for 6 h. The data are presented as means ± sd (n = 3). Significant difference was determined by Tukey test and labeled with different letters (P < 0.05).

DISCUSSION

OsCASP1 Is Required for CS Formation at Endodermis of Rice Roots

Although there are five genes encoding CS domain proteins (OsCASP1 to 5) in the rice genome, we found that only OsCASP1 is required for CS formation at the endodermal cells in rice root (Figure 3; Supplemental Figures 3, 8, and 13). This is in agreement with the expression patterns of these genes; only OsCASP1 is highly expressed in the roots (Figure 1A; Supplemental Table 1). OsCASP1 initially showed a uniform localization at the plasma membrane of the endodermal cells of the root tip without CS (Figures 2A and 2E; Supplemental Figure 4), followed by localization at the middle of anticlinal side of endodermis cells in the mature root region where the CS is formed (Figures 2F and 2G; Supplemental Figure 4). The high expression of OsCASP1 was only found in the root elongation zone (3 to 6 mm from the root apex), while OsCASP1 was also observed in the mature root region (Figure 2H). This result suggests that once OsCASP1 is synthesized at the elongation zone, it will barely be degraded as the root elongates.

Rice roots have one CS at the exodermis and one CS at the endodermis (Enstone et al., 2002). Knockout of OsCASP1 did not affect CS formation at the exodermis (Supplemental Figure 7), suggesting that CS formation in the exodermis is controlled by different unknown genes. Knockout of OsCASP1 resulted in penetration of PI into the stele in the root tip zone (<10 mm from the root apex; Figures 3E to 3J), indicating that the CS as a diffusion barrier at the endodermis is defective in the mutants. Therefore, we supposed that similar to AtCASPs, OsCASP1 plays an important role in CS formation through forming a protein scaffold at the CS domain. However, knockout of AtCASP1 alone did not affect CS formation in Arabidopsis and only the atcasp1 atcasp3 double mutant yielded obvious defects in CS formation (Roppolo et al., 2011), which showed extensive ectopic lignin deposition in the corners of the endodermal cells. Compared with the atcasp1 atcasp3 double mutant, ectopic deposition of lignin at the endodermis in oscasp1 mutants appears less severe. In contrast to the wild type, where a fine band in the anticlinal side of the endodermal cell wall was formed, broad sheet-like lignification was observed in the mutants (Figure 3).

Recently, the CIF1/2-SGN3-SGN1 signal pathway was identified to function in the surveillance of CS integrity and activation of compensatory lignification in response to CS damage in Arabidopsis (Pfister et al., 2014; Doblas et al., 2017; Nakayama et al., 2017). This signal pathway is composed of peptide ligands (CIF1 and CIF2), peptide receptor SNG3, and its downstream target SNG1. A database search using the amino acid sequences of CIF1, CIF2, SGN3, and SGN1 showed that their homologs with high identity (32 to 66%) exist in the rice genome. However, it remains to be determined whether a similar pathway exists in rice.

OsCASP1 Plays a Distinct Role in Controlling Mineral Element Uptake

Knockout of OsCASP1 resulted in a significant reduction in growth when plants were grown in nutrient solution and soil (both upland and flooded conditions; Figure 4; Supplemental Figure 11A and 11B). This is different from Arabidopsis mutants defective in CS formation. For example, obvious growth defects were not observed in atcasp1 or atcasp1 atcasp3 (Roppolo et al., 2011). These differences indicate that, unlike in Arabidopsis, CS formation in rice roots is required for healthy growth.

Remarkable differences were found in the ionomic phenotype between CS mutants of rice and Arabidopsis. In Arabidopsis mutants, including the atcasp1atcasp3 double mutant, esb1, myb36, and lotr1, lower concentrations of Mg, Ca, Mn, and Fe and a higher level of K, S, and Mo in the shoots were found, although there are some discrepancies between different studies (Hosmani et al., 2013; Kamiya et al., 2015; Li et al., 2017). In the sgn3 mutant, the K concentration in the shoots was significantly lower compared with the wild type (Pfister et al., 2014). However, oscasp1 showed a higher concentration of Ca, Sr, and P and a lower concentration of As, Cd, Fe, Ge, Mn, and Zn in the shoots (Figures 5A and 5B). The concentration of Mg, K, Rb, and Cu in the shoots was hardly altered in the mutant lines (Figure 5C). These differences could be attributed to the root structure and uptake system. Rice roots have one CS at the exodermis and one CS at the endodermis (Supplemental Figure 15). Elemental ions move radially through the roots via a combination of apoplastic, symplastic, and transcellular pathways (Steudle, 2000; Geldner, 2013), and their contribution depends on different ions. The apoplastic pathway is blocked by hydrophobic barriers in both the exodermis and endodermis, while the latter two pathways require elemental ion to cross plasma membranes at least twice through polarly localized influx and efflux transporters (Sasaki et al., 2016; Che et al., 2018). For example, in contrast to Arabidopsis roots, the characteristic feature of rice roots is active Si uptake, which is mediated by Lsi1 (influx) and Lsi2 (efflux) transporters localized at the distal side and proximal side of the exodermis and endodermis where CS is formed (Ma et al., 2006, 2007). Knockout of OsCASP1 resulted in a significant decrease in Si and Ge uptake (Figures 5A and 6B). We found that this decrease is not caused by changing polarity of Lsi1 but by decreasing Lsi1 protein abundance. Mathematical modeling has shown that the double-layer structure of the CSs plays an important role in Si transport by preventing backflow of Si from the stele (Sakurai et al., 2015). This study showed that CS is also important for maintaining high Lsi1 protein levels at the endodermis (Supplemental Figure 15). Since arsenite transport is also mediated by Lsi1, this also therefore resulted in a decrease in As accumulation in the shoots (Figure 5B). Recently, Wang et al. (2019) reported that loss of CS integrity was sensed by the CIF1/2-SGN3-SGN1 signal pathway, resulting in the inactivation of aquaporins responsible for reducing root hydraulic conductivity in Arabidopsis. Lsi1 belongs to the aquaporin family (Ma et al., 2006). It is possible that the reduction of Lsi1 protein abundance in oscasp1 was caused by a similar mechanism triggered by the CIF1/2-SGN3-SGN1 signal pathway, although the exact mechanism for this requires further investigation.

Different from Arabidopsis, rice is a Mn-accumulating plant, and its uptake is mediated by a cooperative system formed by OsNramp5 (influx) and OsMTP9 (efflux) polarly localized at the exodermis and endodermis (Sasaki et al., 2012; Ueno et al., 2015; Shao et al., 2017). Similar to Si, decreased Mn accumulation in the shoots of oscasp1 is probably caused by affecting transporter protein abundance (Supplemental Figure 15). Decreased Cd accumulation may also be due to this change in transporter protein abundance, because OsNramp5 is also a major transporter for Cd (Sasaki et al., 2012). Therefore, reduced growth of the oscasp1 mutants is a result of a disrupted transport system of mineral elements in rice, especially Ca, as discussed below.

CS Plays an Important Role in Controlling Apoplastic Ca Movement in Rice Roots

The contrasting result between rice and Arabidopsis mutants defective in CS formation is in the Ca accumulation in the shoots. In all Arabidopsis CS mutants, a decreased Ca accumulation in the shoots was found (Hosmani et al., 2013; Kamiya et al., 2015; Li et al., 2017). By contrast, rice oscasp1 showed an elevated Ca concentration in the shoots when grown under all conditions (Figures 5A and 7G; Supplemental Figures 11D and 11E), suggesting that a defect of CS formation increases Ca uptake in rice. This is also supported by a 1-d exposure to Sr, a chemical analog of Ca, and apoplastic tracer (Figure 5A; Kuppelwieser and Feller, 1991). The difference in Ca accumulation between CASP1 mutants of rice and Arabidopsis could be attributed to the uptake site of Ca due to differences in the root structure. In rice, it seems that apoplastic Ca uptake mainly occurs in the root tip (within 10 mm of the root apex), where suberin lamellae and CS at the exodermis are not completely formed (Figure 3; Supplemental Figures 10 and 15), but the CS at the endodermis has been formed (Figure 3). Therefore, knockout of CASP1 loses the control of Ca movement through the endodermis, resulting in overaccumulation of Ca in the leaves and subsequently growth inhibition (Figures 7A to 7F; Supplemental Figures 14 and 15). However, at the mature root region (>15 mm), since CS at the exodermis is fully formed (Supplemental Figure 15), Ca is largely blocked at the exodermis and not able to enter the root apoplast and reach the endodermis. Therefore, although suberin lamellae formation at the endodermis is accelerated in the mature root region of the mutant roots (Supplemental Figures 10 and 15), it will not affect Ca uptake. This result is also supported by the results of PI penetration at different root regions (Figures 3E to 3M). By contrast, since Arabidopsis roots lack the exodermis, Ca is able to enter the root apoplast and reach the endodermis in the whole roots. Therefore, ectopic suberin deposition at the endodermis in Arabidopsis mutants defective in CS formation inhibits Ca flow and decreases Ca accumulation in the shoots (Attia et al., 2008; Barberon et al., 2016; Li et al., 2017). These spatial differences in the root structure underlie the different effects of CASP knockout on Ca accumulation in rice and Arabidopsis (Supplemental Figure 15).

In conclusion, OsCASP1 is required for CS formation at the endodermis of rice roots. Through functional analysis of oscasp1 mutants, we have revealed that, in contrast to Arabidopsis, the rice CS has a distinct effect on root selective uptake of mineral elements, especially of Ca and Si.

METHODS

Plant Materials and Growth Conditions

Wild-type rice (Oryza sativa; cv Nipponbare) and the knockout lines of five OsCASP genes were used in this study. All the seeds were soaked in deionized water for 2 d in the dark at 28°C and then placed on a net floating on a solution containing 0.5 mM CaCl2. After growth for 4 to 7 d at 28°C, seedlings were transferred to a 4.0-L plastic pot containing one-half-strength Kimura B solution (pH 5.6) as described previously by Yamaji and Ma, (2007) for various experiments. The nutrient solution was changed every 2 d. The plants were grown in a greenhouse under natural light conditions at 25 to 30°C. At least three biological replicates were made for each experiment.

Generation of Five OsCASPs Knockout Lines

To create the knockout lines of five OsCASP genes, the CRISPR/Cas9 genome-targeting system was used. The pCRISPR-OsCASPs plasmids with OsCASPs-specific target sites (the target sequences are shown in Supplemental Table 2) were constructed as described previously by Ma et al., (2015). In brief, specific target sequences within five OsCASP genes were selected by a Blast search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of the target sequences including protospacer adjacent motif sequence against the rice genome sequence. Then target sequences were introduced into single guide RNA (sgRNA) expression cassettes by overlapping PCR with four primers for each OsCASP gene (primer sequences were shown in Supplemental Table 2), producing pU6a-OsCASPs-sgRNA or pU3-OsCASPs-sgRNA fragments. Using BsaI, these fragments were cloned into pYLCRISPR/Cas9Pubi, resulting in the pCRISPR-OsCASPs constructs. These resulting constructs were introduced into Agrobacterium tumefaciens strain EHA101 and transformed into the wild-type rice (cv. Nipponbare for oscasp1, oscasp2, oscasp3, oscasp4, and cv Zhonghua 11 for oscasp5). The homozygous mutants were screened by PCR using primer pairs (primer sequences are shown in Supplemental Table 2) flanking the OsCASPs-specific target sites. The knockout lines of five OsCASP genes were selected for further phenotypic analysis as described below.

Complementation Test

For the complementation test of oscasp1-1, we amplified a 3.478-kb genomic DNA fragment containing the 2.082-kb OsCASP1 promoter sequence, coding sequences of OsCASP1 and the 0.527-kb downstream sequence (after the translational stop site) from wild-type genomic DNA by a PCR approach with primer sets POsCASP1-F and UOsCASP1-R (Supplemental Table 2). The amplified fragment was inserted into a binary vector, pCAMBIA1300. This construct was transformed into calluses derived from the oscasp1-1 mutant via Agrobacterium strains EHA101. Growth at the flooded soil was compared among the wild-type line, oscasp1 mutant, and two independent transgenic lines.

RNA Extraction and Gene Expression Analysis

To investigate the expression pattern of five OsCASP genes, we sampled various tissues including root, leaf blade, leaf sheath, and spike at the heading stage. The spatial expression of OsCASP1 was examined using different segments (0 to 3, 3 to 6, 6 to 9, 9 to 12, and 12 to 15 mm from root apex) of 5-d-old seedlings. To compare the expression of Lsi1 between the wild type and oscasp1 mutants, different segments (0 to 6, 6 to 12, and 12 to 18 mm from root apex) of 5-d-old seedlings of both the wild type and oscasp1 mutants were sampled for RNA extraction. Total RNA was extracted using a Trizol reagent kit (Life Technologies). Total RNA (1 μg) was used for first-strand cDNA synthesis using the PrimeScript II 1st Strand cDNA Synthesis kit (Takara), following the manufacturer’s instructions. The expression was determined with SYBR Premix ExTaq II (Takara) by qTOWER 2.0 (Analytik Jena). Three biological replicates were performed for each experiment. Histone H3 was used as an internal standard. The primer sequences are listed in Supplemental Table 2.

OsCASP1-GFP Transgenic Rice

To examine the cellular localization of OsCASP1, we generated transgenic plants carrying the pOsCASP1-OsCASP1-GFP fusion. The 2.082-kb native promoter of OsCASP1 was amplified from rice (cv Nipponbare) genomic DNA with the primer sets POsCASP1-GFP-F and POsCASP1-atg-R (Supplemental Table 2), and OsCASP1 was amplified from Nipponbare cDNA of the roots with the primer sets POsCASP1-atg-F and POsCASP1-GFP-R. Using an in-Fusion HD cloning kit (Takara), the amplified fragments were inserted into pCAMBIA1301-GFP, producing the ProOsCASP1-OsCASP1-GFP construct. This construct was transformed into calluses derived from Nipponbare via Agrobacterium strains EHA101. The primer sequences are listed in Supplemental Table 2.

To examine whether pOsCASP1-OsCASP1-GFP is functional, we crossed the oscasp1 mutant with a transgenic line carrying pOsCASP1-OsCASP1-GFP. Among the F2 population, we selected homozygous oscasp1 lines carrying the ProOsCASP1-OsCASP1-GFP or not using primers 5′-gcc​att​cag​gct​gcg​caa​ctg-3′ and 5′-ggt​agc​aga​ttt​ggt​gtt​ag-3′. The wild type, selected F2 lines carrying pOsCASP1-OsCASP1-GFP or not were grown in a 6.0-L pot containing soil collected from Nanning, Guangxi province, China. After 3 and a half months, the plants were photographed.

Subcellular Localization

To construct the OsCASP1-GFP fusion protein, the OsCASP1 coding sequence was amplified with primer set OsCASP1-GFP-F and OsCASP1-GFP-R (Supplemental Table 2). Using EcoRI and SalI, the amplified fragment was cloned into the vector pYL322-d1-eGFP containing a GFP gene driven by the 35S promoter. To construct an mCherry-OsRac3 fusion, a plasma membrane marker (Chen et al., 2010), the open reading frame of OsRac3 was amplified from the Nipponbare cDNA with primer set CDS-OsRac3 (Supplemental Table 2). The resulting fragment was inserted in frame after the mCherry coding region into the p35S-mCherry-NosT vector. Rice protoplast preparation and plasmid transfection were performed as described previously by Chang et al., (2018). After transformation, cells were incubated at 28°C in darkness for 12 to 15 h. The fluorescence images were taken using a confocal laser scanning microscope (TCS SP8; Leica Microsystems). The primer sequences are listed in Supplemental Table 2.

Immunostaining and Lignin/Cellulose Staining

To observe the localization of OsCASP1-GFP and Lsi1 in the roots, immunostaining was performed using an antibody against GFP (1:1000 dilution; GFP polyclonal antibody A-11122; Thermo Fisher Scientific) or Lsi1 (1:500 dilution; Ma et al., 2006) as described previously by Yamaji and Ma, (2007). Root cross sections (100 µm thickness) were prepared using a microslicer (Linear Slicer PRO10; Dosaka EM). Before the last wash step of the immunostaining, lignin and cellulose were stained with 0.2% (w/v) Basic Fuchsin and 0.1% (w/v) Calcofluor White in PBS buffer, respectively, according to Ursache et al. (2018) without ClearSee. Lignin/cellulose staining was performed similarly in all OsCASP knockout lines, but without antibody treatments. Fluorescence was observed with a confocal laser-scanning microscope (TCS SP8x; Leica Microsystems). For clear observation of the strip-shape localization of CSs and OsCASP1-GFP, z-stack magnified images of root inner tissues were taken (∼20 optical sections with 0.3∼0.4 µm intervals for each sample), and the 3D images were constructed by LAS X 3D software (Leica Microsystems). To image Basic Fuchsin, an excitation of 580 nm was used, and the signal was detected at 600 to 615 nm. To image Calcofluor White, an excitation of 405 nm was used and the signal was detected at 415 to 440 nm. To image secondary antibody labeled with Alexa Fluor 488, an excitation of 500 nm was used and the signal was detected at 510 to 525 nm.

PI Staining

A PI penetration assay was performed as previously described (Alassimone et al., 2010). Eight-day-old seedlings were exposed to the solution with 15 µg/mL PI (P4170; Sigma) in the dark at 30°C for 1 h. After rinsing with water, the roots were embedded in 5% (w/v) agar and sectioned at different positions (10 and 20 mm from the apex) with a slicer (Linear Slicer PRO10; Dosaka EM). Sections (100 μm thickness) were mounted with 50% (v/v) glycerol for observation with a laser-scanning confocal microscope (TCS SP8x, Leica Microsystems). To image PI, an excitation of 405 nm was used, and the signal was detected at 600 to 650 nm. For a shorter exposure, 5-d-old seedlings were exposed to PI in the dark for 40 min at 28°C. Other procedures were as described above.

Suberin Staining

Suberin staining was performed with Fluorol Yellow 088 according to Lux et al. (2005). Root cross sections at different root positions (15 and 30 mm from the root apex) were incubated in a freshly prepared solution of Fluorol Yellow 088 (Sigma; 0.01% [w/v] in lactic acid) at 70°C for 30 min. The specimens were then rinsed in water three times at 70°C, placed on glass slide, and observed by confocal scanning microscopy (TCS SP8x, Leica Microsystems). To image Fluorol Yellow 088, an excitation of 405 nm was used, and the signal was detected at 510 to 525 nm.

Phenotypic Analysis of the Knockout Lines

To observe the phenotype of OsCAPS1 mutants, we grew both wild-type rice and two mutants in nutrient solution and soil. Seedlings (12-d-old) prepared as described above were grown in 1.2-liter pots containing half strength Kimura B solution with three biological replicates. The nutrient solution was changed once every 2 d. The plants were grown for 34 d. Before harvest, the plants were exposed to a solution containing 1 µm of Sr, Rb, and Ge, and 0.2 µm of As and Cd for 1 d. The roots were washed with cold water three times and separated from the shoots for mineral analysis as described below.

For soil culture, seedlings (12-d-old) of both wild-type rice and the knockout lines prepared as described above were transplanted to a 3.5-L plastic pot containing paddy soil collected from a farm at the Institute of Plant Science and Resources, Okayama University. Soil was kept at upland or flooded conditions with tap water. After 45 d, the shoots were harvested for mineral element analysis as described below.

The seedlings of wild-type rice and five oscasp mutants were also grown under flooded conditions in a 6.0-L pot containing soil collected from Nanning, Guangxi province, China. The growth was observed at the vegetative and reproductive growth stage. At harvest, the growth parameters were investigated, and the plants were photographed.

To compare the ionomes between other oscasp mutants and their wild-types, seedlings (4 weeks old) prepared as above were exposed to a nutrient solution containing 5 mm CaCl2 for 10 d. The solution was changed every 2 d. Four replicates were made for each line. Before harvest, the plants were exposed to a solution containing 1 µm of Sr, Rb, and Ge and 0.2 µm of As and Cd for 1 d as described above.

Time Course Experiments of Si and Water Uptake

The time-dependent uptake of Si was investigated with both wild-type rice and two knockout lines according to the procedures described in Ma et al. (2002). In brief, seedlings (52-d-old) were placed in a 50-mL black bottle containing one-half-strength Kimura B solution (pH 5.6) with 1.0 mm Si as silicic acid. At the time points indicated in Figure 6, a 0.5-mL aliquot of uptake solution was taken to determine the Si concentration. Water uptake (water loss) was also recorded at each sampling time. At the conclusion of the experiment, the roots were harvested and the fresh and dry weights were recorded.

Growth at Different Ca Concentrations

To investigate the effect of different concentrations of Ca on growth, two knockout lines and the wild type (2 weeks old) were grown in one-half-strength Kimura B solution containing different Ca concentrations (0.18, 1, 5, and 10 mm) as CaCl2. After 12 d, the plants were photographed. The roots and shoots were separately collected with a razor and subjected to Ca determination as described below.

Collection of Xylem Sap

Seedlings (30 d old) were exposed to one-half-strength Kimura B solution containing different Ca concentrations (0.18, 1, 5, and 10 mm). After 6 h, the shoots (2 cm above the roots) were excised with a razor, and then the xylem sap was collected with a micropipette for 1 h after decapitation of the shoot. The Ca concentration of the xylem sap was determined as described below after being diluted with 2% HNO3.

Elemental Analysis of Plant Tissues

All the samples were dried at 70°C for 3 d. The dried samples were digested with 65% HNO3 at a temperature of up to 140°C. The metal concentration in the xylem sap and the digested solution was determined by inductively coupled plasma-mass spectrometly (Plasma Quant MS Elite; Analytik Jena AG or ICP-MS 7700X; Agilent Technologies).

Accession Numbers

Sequence data from this article can be found in the DDBJ/GenBank/EMBL data libraries under accession numbers OsCASP1, LOC_4337453 (Os04g0684300); OsCASP2, Os08g0101900; OsCASP3, Os06g0231050; OsCASP4, Os02g0743900; OsCASP5, Os04g0460400.

Supplemental Data

DIVE Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

Acknowledgments

This work was supported by the Grant-in-Aid for Specially Promoted Research (JSPS KAKENHI grant 16H06296 to J.F.M.), the National Natural Science Foundation of China (31670253), the Guangxi Natural Science Foundation (2016GXNSFFA380013), a Guangxi innovation-driven development special funding project (grant Guike-AA17204070), and the Project of High Level Innovation Team and Outstanding Scholar in Guangxi Colleges and Universities (2016).

AUTHOR CONTRIBUTIONS

J.F.M. and J.X. designed the research. Z.W., N.Y., and S.H. equally contributed to the research. Z.W., N.Y., X.Z., M.S., S.H., S.F., G.Y., J.F.M., and J.X. performed the experiments. Z.W., N.Y., S.H., J.F.M., and J.X. analyzed data. J.F.M. and J.X. wrote the article.

Footnotes

  • www.plantcell.org/cgi/doi/10.1105/tpc.19.00296

  • The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Jian Feng Ma (maj{at}rib.okayama-u.ac.jp), Jixing Xia (xiajx{at}gxu.edu.cn).

  • ↵1 These authors contributed equally to this work.

  • Received April 25, 2019.
  • Revised August 16, 2019.
  • Accepted August 31, 2019.
  • Published September 4, 2019.

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OsCASP1 Is Required for Casparian Strip Formation at Endodermal Cells of Rice Roots for Selective Uptake of Mineral Elements
Zhigang Wang, Naoki Yamaji, Sheng Huang, Xiang Zhang, Mingxing Shi, Shan Fu, Guangzhe Yang, Jian Feng Ma, Jixing Xia
The Plant Cell Nov 2019, 31 (11) 2636-2648; DOI: 10.1105/tpc.19.00296
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