CRISPR/Cas9-Engineered Large Fragment Deletion Mutations in Arabidopsis CEP Peptide–Encoding Genes Reveal Their Role in Primary and Lateral Root Formation

Abstract

C-TERMINALLY ENCODED PEPTIDEs (CEPs) are post-translationally modified peptides that play essential roles in root and shoot development, nitrogen absorption, nodule formation and stress resilience. However, it has proven challenging to determine biological activities of CEPs because of difficulties in obtaining loss-of-function mutants for these small genes. To overcome this challenge, we thus assembled a collection of easily detectable large fragment deletion mutants of Arabidopsis CEP genes through the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9–engineered genome editing. This collection was then evaluated for the usability by functionally analyzing the Arabidopsis growth and development with a focus on the root. Most cep mutants displayed developmental defects in primary and lateral roots showing an increased primary root length and an enhanced lateral root number, demonstrating that the genetic resource provides a useful tool for further investigations into the roles of CEPs.

Introduction

Small signaling peptides serve as local or long-distance signal molecules to coordinate plant cell–cell communications in various biological processes (Matsubayashi 2014, Olsson et al. 2019). One such example is the C-TERMINALLY ENCODED PEPTIDE (CEP) family, which plays critical roles in a wide range of physiological and developmental processes as well as stress acclimatization (Taleski et al. 2018). CEP precursor proteins consist of an N-terminal signal peptide sequence, a central variable domain and one or multiple conserved CEP motifs (Taleski et al. 2018). The mature CEP peptides are commonly post-translationally modified CEP motifs containing hydroxylated proline residues. CEP family members are widely present in seed plants including Arabidopsis, in which a total of 15 CEP genes have been identified (Delay et al. 2013, Roberts et al. 2013). Previous studies revealed that Arabidopsis CEP peptides are key regulators in root and shoot development (Ohyama et al. 2008, Delay et al. 2013, Roberts et al. 2013, 2016, Chapman et al. 2019), nitrogen absorption and nodule formation (Imin et al. 2013, Tabata et al. 2014, Mohd-Radzman et al. 2016, Laffont et al. 2020, , Luo et al. 2022) and stress resilience (Delay et al. 2013, Delay et al., 2019, Smith et al. 2020). Despite these advances, only a very few Arabidopsis CEP family members have defined functions. The functional role of the majority of CEP peptides, however, remains largely unknown.

It has proven challenging to determine biological activities of CEP peptides. One major barrier in elucidating the role of CEP genes is the lack of loss-of-function mutants, which are hardly acquired by canonical mutagenesis methods like transfer DNA (T-DNA) tagging, largely due to the fact that these small genes encoding a short stretch of amino acids are less likely to be targeted by a T-DNA insertion. In addition, gain-of-function studies via overexpression or exogenous peptide application caused similarly pleiotropic phenotypes that were the integrated results of hypermorphic and neomorphic effects upon abundant ectopic ligands through multiple signaling pathways, which may not reflect the endogenous functions of CEP genes. As such, there is an urgent need to generate loss-of-function mutants for further investigations into the role of CEP peptides.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) is an efficient gene editing technique that has been widely applied in plants (, Zhan et al. 2021). Thus, in this study, we assembled a collection of large fragment deletion mutants for CEP peptide–encoding genes by CRISPR/Cas9-mediated gene editing technology. Subsequently, to evaluate the usefulness of these mutants for understanding the CEP-mediating biological activities, this collection was functionally characterized for altered phenotypes in root growth and development as a start. Our analysis of cep mutants clearly revealed altered primary and lateral root phenotypes across all cep mutants, except the cep6 mutant showing no alteration in the primary root. Furthermore, our results clarified the reported roles in root growth and development of CEP3 and CEP5 genes using these knock-out mutants. The assembly of CRISPR/Cas9-mediated cep mutants thus is a valuable source for future investigations into the biological roles of CEPs.

Results and Discussion

To generate easily detectable deletion mutants for CEP genes, we designed two guide RNAs that targeted each CEP gene to generate possible large fragment deletions, which can be easily identified by polymerase chain reaction (PCR). To this end, a cassette that enables mCherry expression under a seed-specific promoter was fused to the CRISPR/Cas9 vector (Xing et al. 2014, Wang et al. 2015, Gao et al. 2016), thus facilitating us to visually select fluorescent mutant seeds of T1 plants and non-fluorescent mutant seeds of next generations to get Cas9-free plants (Supplementary Fig. S1). To completely disrupt CEP peptide–encoding genes, most of the two targets covered the full coding region containing the conserved functional CEP motifs in order to delete the coding sequence and obtain null cep mutants (Fig. 1, Supplementary Table S1). No mutant was generated for CEP9, CEP10 and CEP11 genes after several attempts due to unknown reasons. Thus, we successfully generated 20 mutant alleles containing deletions spanning from 100 to 400 bp for all remaining 12 CEP genes (Fig. 1). Among them, two independent mutant alleles are produced for CEP1, CEP2, CEP3, CEP4, CEP5, CEP8, CEP13 and CEP15 genes (Fig. 1). In few cases, several base insertions were also observed (Fig. 1). Subsequently, the stable and heritable CRISPR/Cas9 vector–free cep alleles were obtained for functional analysis. As such, each mutant harbored a large fragment deletion covering the coding region or the functional CEP motifs, thus resulting in null alleles.

Next, we evaluated our collection of cep mutants to examine whether the collection could be a useful tool for the functional analysis of CEP peptides in Arabidopsis. To this end, we examined the phenotypes of the complete collection of cep mutants with respect to their growth and development. In comparison to wild-type (WT) plants, no significant difference was observed in all cep mutants regarding their overall growth and development, whereas the aboveground and/or underground parts in some cep seedling mutants were greater. Thus, the fresh weight of shoots and roots was measured across all cep mutants. In comparison to WT shoots, the shoot fresh weight of cep2-c1, cep2-c2, cep4-c1, cep4-c2, cep7-c1, cep8-c1 and cep8-c2 was significantly increased (Fig. 2). A substantial proportion of cep mutants, other than cep14-c1, cep15-c1 and cep15-c2, exhibited a significantly greater root fresh weight than that of WT controls (Fig. 2), implying that many CEP peptides are implicated in the root growth and development. While cep14-c1, cep15-c1 and cep15-c2 mutants showed a greater root fresh weight, no significance is observed (Fig. 2). Altogether, these results indicate that many CEP peptides are negative regulators with respect to shoot and/or root growth and development.

Given the prominent roles of CEP peptides in root development (Fig. 2, Taleski et al. 2018, Olsson et al. 2019), we further assessed the role of CEP genes in primary root and lateral root development. Intriguingly, all cep mutants, other than cep6-c1, exhibited significantly longer primary roots compared to WT seedlings (Fig. 3) consistent with CEP overexpression or exogenous application of synthetic CEP peptides, which both caused shorter primary roots (Ohyama et al. 2008, Delay et al. 2013, Roberts et al. 2013, 2016, Delay et al., 2019). A close inspection of the meristematic zone indicated that the longer primary root length could be explained by the enlarged root meristem size observed in cep mutants (Fig. 4). Previous studies indicated that CEP1, CEP5, CEP6, CEP8, CEP9 and CEP15 were expressed in primary roots (Roberts et al. 2013, 2016, Tabata et al. 2014). The public expression data are available for CEP1, CEP3, CEP5, CEP9, CEP12, CEP13 and CEP15, all of which were found to be expressed in primary roots (Supplementary Fig. S2). The expression confirms the role of these CEP genes in the regulation of the primary root. Although CEP6 is highly expressed in primary roots (Tabata et al. 2014), the root of the cep6-c1 mutant is indistinguishable from that of WT plants (Figs. 3, 4), indicating that CEP6 might be dispensable for primary root growth. Alternatively, the CEP6 peptide may function redundantly with other CEPs in the regulation of primary root development. Nevertheless, additional mutant alleles for the CEP6 gene are needed to clarify its function in the primary root growth. It was reported previously that CEP3 is a negative regulator in primary root growth on the observation that a T-DNA insertion mutant, cep3-1, displayed a longer primary root and an increased meristem size under nitrogen-scarce conditions, but not under nitrogen-sufficient conditions (Delay et al. 2013, Delay et al., 2019). By contrast, cep3-c1 and cep3-c2 from our collection exhibited an increased root meristem size and a longer primary root length under normal nutritional conditions (Figs. 3, 4). The discrepancy is likely because of differences in the experimental medium; a sucrose-free half-strength Murashige and Skoog (MS) medium was used in Delay et al. (2019). A CEP5 RNA interference (RNAi) line showed a significantly longer primary root length (Roberts et al. 2016). Consistently, our newly generated cep5-c1 and cep5-c2 mutants displayed an increased primary root length and an enlarged meristem size (Figs. 3, 4), thus strongly indicating a prominent role of CEP5 peptide in primary root meristem regulation. The similar phenotype of increased primary root length and enlarged root meristem size in each single cep mutant implies that less but conceivable overlapping activities exist among CEP peptides, at least in the regulation of the primary root development. Taken together, our data clearly indicate that CEPs play similar and crucial roles in controlling root development.

Previous studies also suggested that CEP peptides are important players in lateral root development either under normal growth conditions (Imin et al. 2013, Mohd-Radzman et al. 2016, Roberts et al. 2016) or under nutrient-limited and stress conditions (Delay et al. 2013, Chapman et al. 2019). We therefore also tested the collection of cep mutants for altered lateral root phenotypes. In line with the increased root fresh weight (Fig. 2), the number and density of lateral roots in all cep mutants were significantly increased in comparison to WT plants (Fig. 5), indicating that CEP peptides play important roles in regulating the formation of lateral roots. Concordantly, exogenously applied CEP peptides or CEP overexpression decreased the lateral root number (Delay et al. 2013, Roberts et al. 2016, Chapman et al. 2019). Consistent with the increased lateral root number of reported CEP5 RNAi lines (Roberts et al. 2016), we found a significant addition of lateral root number in cep5-c1 and cep5-c2 mutants compared to WT plants (Fig. 5). The cep3-1 mutant exhibited an increased lateral root number under nitrogen-limiting conditions due to increased primary root growth (Delay et al. 2013). Moreover, the cep3-1 mutant showed an increased lateral root density under higher osmotic strength, increased irradiance and phosphate- and nitrate-limited conditions (Delay et al. 2013). This is consistent with the finding that the CEP3 synthetic peptide significantly decreased lateral root density (Delay et al. 2013). In the case of cep3-c1 and cep3-c2 mutants, both showed an elevated lateral root number and density under normal growth conditions (Fig. 5), suggesting that both intrinsic and induced CEP signaling pathways corporately modulate the lateral root formation. In addition to the increased lateral root number and density, we found that cep4-c1, cep4-c2, cep8-c1 and cep8-c2 exhibited a significantly longer lateral root (Fig. 5), suggesting that CEP4 and CEP8 peptides may also function in the regulation of lateral root elongation. However, the increase in the lateral root length was not observed in other cep mutants (Fig. 5). The observed phenotypic similarities in lateral root formation and variance in the degree of defects across all cep mutants suggest that their functions could be unequally redundant. Furthermore, our results indicate that, in addition to nutrient-deficient and stress-regulated Arabidopsis lateral root development (Delay et al. 2013, Chapman et al. 2019), CEP peptides appear to also modulate lateral root growth independent of nutritional conditions in Arabidopsis.

Although many efforts have been made to unravel the biological roles of CEP peptides, to date, a function has been assigned only to a very limited number of CEP peptides mainly on the results of gain-of-function studies (Taleski et al. 2018, Olsson et al. 2019). In this study, we assembled a collection of CRISPR/Cas9-engineered mutants for Arabidopsis CEP genes to meet the urgent need for unraveling CEP-mediated signaling pathways (Fig. 1). A total of 20 mutant alleles were generated for 12 out of all 15 CEP genes. Therefore, the generation of mutant alleles for the remaining CEP9, CEP10 and CEP11 genes is still required. To fill in this gap, available T-DNA insertion alleles are obtained from the public collections (O’Malley et al. 2015).

By using the cep mutant collection, we performed a functional analysis of CEP peptides with an emphasis on their primary and lateral root growth and development to validate the usefulness of this mutant collection in understanding the CEP-mediating biological activities. Intriguingly, the majority of cep mutants, except the cep6 mutant, exhibited a longer primary root length and an enlarged primary root meristem size (Figs. 3, 4), and all cep mutants displayed an increased density and number of lateral roots (Fig. 5). Nevertheless, the spatiotemporal expression of CEP genes using transcriptional reporters will contribute to a better understanding of the role of CEP peptides in plant growth and development. Moreover, the underlying mechanisms of CEP-regulated root meristem size and lateral root formation remain to be investigated. Our results suggest that, contrary to previous speculation, less and unequal functional redundancy seems to exist among CEP peptides. However, the overlapping activities of CEP genes through the generation of high-order mutants by combining these cep mutants are yet to be examined, the highly conversed functional CEP motif harbored in each CEP precursor. In addition, it will be intriguing to determine new biological activities of CEP peptides by performing an extensive examination using our cep mutant collection with respect to a broader range of plant growth and development and sensitivity to various stress responses. Collectively, the assembly of CRISPR/Cas9-mediated cep mutants provides a valuable source for future investigations into the biological roles of CEPs.

Materials and Methods

Plant materials and growing conditions

WT seeds of Arabidopsis Columbia-0 (Col-0) and cep mutant seeds were surface-sterilized and sown in 1/2 MS medium containing 1% sucrose. Plants were grown in growth chambers with a long-day condition (16-h light and 8-h darkness) at 22°C.

Vector construction and transformation

The CRISPR/Cas9 vector pHEE401E-mCherry is a generous gift from Prof. Yunde Zhao (University of California San Diego). The pHEE401E-mCherry vector was generated by fusing the seed-specific At2S3 promoter–driven mCherry to the pHEE401E vector skeleton (Xing et al. 2014, Wang et al. 2015, Gao et al. 2016). Target sequences were designed manually with the aid of the CRISPRdirect (Naito et al. 2015). High-scoring target sequences were then retrieved, and the off-target potential was evaluated using Rgenome. Primer Premier 5.0 was used for primer selection, and SnapGene and DNAMAN 6.0 were used for sequence analysis. Primers containing target sequences were used to amplify dual target elements, and the resulting PCR products were cloned into pHEE401E-mCherry vectors by Gibson reactions (Gibson et al. 2008). The primers used are listed in Supplementary Table S1.

Genotyping analysis

The CRISPR/Cas9 constructs were transformed into Arabidopsis Col-0 plants through floral dipping (Clough and Bent 1998). T1 plants were selected based on seeds showing strong red fluorescence using a dissecting fluorescence microscope equipped with an mCherry filter. To confirm the mutagenesis, selected seeds were planted for DNA extraction and subsequent PCR genotyping. The primers used for genotyping are listed in Supplementary Table S1. CRISPR/Cas9 vector–free lines with no fluorescence were identified for further analysis.

Phenotypic analysis

For the primary and lateral root length and lateral root number measurements, seedlings were grown under long-day conditions (16-h light and 8-h darkness) on vertical plates for 6 and 11 d, respectively. The plates were scanned, and the primary and lateral root length and lateral root number were quantified using Image J. The root meristem size was visualized as previously described (Yang et al. 2017). The lateral root density was determined as the total number of emerged lateral roots/total primary root length. Root meristem images were captured using a ZEISS Axio Scope.A1 microscope equipped with Nomarski differential interference contrast optics. WT plants and cep mutants were grown on standard 1/2 MS medium containing 1% sucrose for 12 d before the shoot and root fresh weight was examined. All phenotypic assays were replicated at least three times.

Supplementary Data

Supplementary data are available at PCP online.

Data Availability

Data and materials are available upon request.

Funding

Shaanxi Provincial Natural Science Foundation (2020JM-284, 2023-JC-ZD-09); National Natural Science Foundation of China (31771556); Natural Science Basic Research Program of Shaanxi for Distinguished Young Scholars (2020JC-29); Fundamental Research Funds for the Central Universities (GK202002008).

Acknowledgements

We thank Prof. Yunde Zhao (University of California San Diego) for providing the CRISPR/Cas9 vector and Dr. Jilin Chen (Institute of Crop Sciences, Chinese Academy of Agricultural Sciences) and Dr. Lejun Ouyang (Guangdong University of Petrochemical Technology) for helping on vector construction.

Author Contributions

G.W. and A.H. conceived, designed and supervised this study. A.H., T.C., Ya.Zh., X.R., M.W., L.J. and Yo.Zh. performed the experiments. G.W. and A.H. wrote the paper with inputs from all authors.

Disclosures

The authors have no conflicts of interest to declare.

References

Author notes