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From an EMS cucumber mutant library, a micro-plant (mp) mutant was identified showing miniaturized phenotypes in all organs (Fig. 1a−g). The plant height of mp mutant shrank significantly by ~80% of normal cucumber plant (CCMC) at the expanded 10 true-leaf stage. Other organs such as hypocotyl, internode length, leaf size, ovary size and fruit length decreased by about 50% to 80% compared with that of CCMC (Supplemental Fig. S1). The mp mutant was female sterile hence unable to produce viable seeds. Specifically, there were fewer seeds in mature fruit of mp mutant, and all of them were empty, deflated and unable to germinate. The hybrid F1 of CCMC and mp mutant displayed an intermediate phenotype, and its growth state was between CCMC and mp mutant (Fig. 1). In addition, the mutant also exhibited significant reduction of several key photosynthetic parameters (Pn, Gs, E and Fv/Fm) in mp mutant than CCMC (Supplemental Table S2).
Micro-structure of stem epidermal cells in mp mutant displayed irregular cell size and deformed cell shape compared with normal cucumber (Fig. 2). Triangle, trapezoid, or sub-circular cells were observed in mp mutant, while only long rhombus cells were observed in the normal plants (Fig. 2a, 2b). The length of epidermal cells in mp stem was significantly shorter than that of CCMC stem, while the width did not change significantly (Fig. 2c, 2d). In addition, the cell number of epidermal cells in mp stem was significantly increased (Fig. 2e). Paraffin sections of the ovary cells of mp mutant and CCMC were also examined, and the result showed that the ovary cells of mutant were smaller and irregularly arranged compared with CCMC (Supplemental Fig. S2). According to these findings, the emergence of micro plant phenotype was caused by changes in cell development and proliferation.
Figure 2.
Scanning electron microscopy (SEM) observation of stem epidermal cells of CCMC and mp mutant. Micrographs of stem epidermal cells in (a) mp mutant and (b) CCMC. Scale bar = 100 μm. Boxplot indicating the (c) length, (d) width and (e) cell number of epidermal cells on stems of CCMC and mp mutant.
Genetic mapping placed the mp locus to a 130.9 kb interval
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To explore the inheritance of mp phenotype, we developed F2 population consisting of 485 plants derived from the cross between CCMC and mp mutant. 117, 255 and 113 plant individuals exhibited micro-plant type, intermediate type and normal plant phenotype in the F2 population respectively, which fits the segregation ratio of 1:2:1 (P = 0.51). This suggested that the micro-plant mutant phenotype is controlled by a single gene with incomplete dominance.
From the F2 plants, we bulked plants with extreme micro-plant and normal-plant phenotypes for re-sequencing. Each pool has a sequencing depth of over 82.26 and coverage of over 93.43% (Supplemental Table S3). The potential polymorphisms were found by aligning the short reads of two bulks to CCMC consensus assembly. The calculated (SNP index) throughout the majority of the genome areas was 0.5, indicating that there was no substantial genetic variation between the two groups ( Fig. 3a ). A single genomic region harboring a cluster of SNPs with a high SNP index was identified in the 15.5−17.6 Mb interval of chromosome 7, suggesting that this candidate genomic region probably harbors the causative mutation ( Fig. 3a).
Figure 3.
BSA-seq and linkage analysis of the mp locus. (a) BSA-seq analysis identified the candidate interval for mp locus to a 2.1 Mb genomic region harboring a high ΔSNP index (subtracting the SNP-index value of the mutant-pool from the CCMC-pool) on chromosome 7 (15.5−17.6 Mb). (b) A genetic map based on a F2 segregation population containing 273 individual plants delimited the mp locus to a 1.1 cM region. (c) Seven polymorphic markers and nine recombinants were applied to narrow down the mp locus to a 130.9 kb region. The numbers in parentheses indicate the number of recombinant plants of each marker. The white box indicates the mutant genotype, the black box for the CCMC genotype, and the striped box for the heterozygous genotype. (d) Genes (black boxes) and candidate mutation site in the mapping region.
For the low level of polymorphism between mp and CCMC at the candidate region, we developed another F2 population (n = 273) from the cross between 'Hazerd' variety and mp mutant to narrow the potential region of mp locus. Ten polymorphic InDel markers and one SNP marker were developed between 'Hazerd' and mp mutant. After the marker genotypes of all 273 individuals were identified, an initial linkage map was constructed and mp locus was mapped to a 0.6 cM region flanking by two Indel markers (MIndel-23 and MIndel-10) (Fig. 3b). To identify more recombinants in this region, a larger F2 population with 708 plants was examined, and two additional SNP markers were developed. Nine more recombinants were obtained, delimiting the mp locus into a 130.9 kb area flanked on Chr7 by markers MSNP-3 (17,480,086 bp) and MIndel-30 (17,611,033 bp) (Fig. 3c).
A gene that encodes the Katanin p60 subunit protein was identified for mp locus
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Nineteen genes were annotated in the 130.9 kb region (Fig. 3d, Supplemental Table S4). Based on the re-sequencing data of CCMC and two bulked pools, five SNPs were found in this region (Supplemental Table S5). Among these SNPs, SNP (SNP-7G17507331) causes a non-synonymous homozygous mutation (Supplemental Table S5). The other four SNPs were located in the intergenic region and had a low SNP index (below 0.6) in mp pool (Supplemental Table S5). There were no other base differences (like Indel or SV) in this region between two bulked pools. As a result, the SNP-7G17507331 was considered as the causal SNP for the mp mutant.
According to the Cucumber Genome Database, SNP-7G17507331 is located inside the gene Csa7G435510 (Fig. 3d). We performed Sanger sequencing to clone the genomic and cDNA sequences of Csa7G435510 from CCMC and mp mutant (Supplemental Fig. S3). The gene Csa7G435510 consists of seven exons and encodes a Katanin p60 protein containing an AAA+-type ATPase domain of amino acid residues 225 to 514 (Fig. 4a, Supplemental Fig. S4). We named it as CsKTN1. The causal SNP at the exon 5 of CsKTN1 resulted in an amino acid substitution from serine (S353) in CCMC to phenylalanine (F353) in mp mutant, which was located at the conserved AAA+-type ATPase domain, suggesting the potential modification in protein function (Fig. 4a, Supplemental Fig. S4).
Figure 4.
Identification of CsKTN1 gene. (a) Diagram of the gene structure of Csa7G435510. A single-nucleotide mutation occurs in the fifth exon of Csa7G435510 resulted in S (Serine) to F (Phenylalanine) substitution in mp mutant. (b) A neighbor-joining tree for cucumber Katanin p60 protein and its homologs with other selected plant species, constructed by MEGA 7. The numbers at the branch points represent bootstrap values (%) of 1000 replications. (c) Relative expression of CsKTN1 in different tissues of mp and CCMC plants measured by qRT-PCR. Values are mean±SD. ** P < 0.01 (Student's t-test). (d) Subcellular localization of CsKTN1 protein (CCMC and mp mutant) in tobacco epidermal cell. GFP signal was observed by confocal fluorescence microscopy. GFP, Bright field and Merged represent the images observed under different light fields. Free-GFP represents the free GFP plasmid p35S::GFP, while CsKTN1F(mp)-GFP and CsKTN1S(CCMC)-GFP represent two KNT1-GFP protein.
To understand the structural and functional relationship among Katanin p60 homologs, we performed protein alignment and constructed a phylogenetic tree for CsKTN1 with 9 Katanin p60 protein sequences from other crops (Supplemental Fig. S4). The alignment showed that AAA+-type ATPase domain is highly conserved across dicot and monocot plants. The similarity of CsKTN1 to other Katanin p60 protein varied from 78% (Arabidopsis homolog) to 93.99% (Melon homolog). The phylogenetic tree showed that CsKTN1 was clustered together with Katanin p60 protein from dicot species and far away from monocot crops (Fig. 4b).
Furthermore, we investigated the gene expression patterns of CsKTN1 in various organs of mp and CCMC, including root, stem, leaf, male flower, ovary and tendril. The result showed that the expression level of CsKTN1 was the highest in stem, followed by root, leaf, flower and tendril, and the lowest in ovary of CCMC. The expression level of CsKTN1 gene was the highest in root, followed by tendril, leaf, stem and flower, and the lowest in ovary of mp mutant. Except for the tendril, the expression of CsKTN1 was significantly lower in all organs of mp mutant compared to the CCMC (Fig. 4c). To characterize the gene function, we examined the subcellular localizations of the protein encoded by two CsKTN1 alleles (CCMC and mp mutant) in tobacco leaf epidermal cells. As a result, both CsKTN1-GFP fusion proteins (CsKTN1S(CCMC)-GFP and CsKTN1F(mp)-GFP) were localized in cell membrane, cytoplasm, and nucleus, similar to free GFP protein (Fig. 4d).
The mutated CsKTN1 protein lost part of its ability to bind and shear microtubule in vitro
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Given that Katanin p60 protein is required for microtubule-shearing in cell[17], the molecular basis for CsKTN1 in regulation of microtubule morphology was investigated in vitro. We applied the coprecipitation assay for two CsKTN1-His fusion proteins (CCMC and mp) to determine whether CsKTN1 could bind to microtubule. As shown in Fig. 5a, in the absence of microtubules, none of the CsKTN1-His proteins was sedimented and retained in the supernatant. After incubation with microtubules, the amount of CsKTN1S (CCMC)-His in the pellets was found more than CsKTN1F (mp mutant)-His, indicating that CsKTN1S (CCMC)-His has a stronger ability to bind and coprecipitate with microtubules (Fig. 5a).
Figure 5.
Microtubule binding and shearing capacity of CsKTN1. (a) Binding of CsKTN1 to microtubules in vitro. The proteins of supernatants (S) and the pellets (P) were analyzed on a coomassie-blue-stained polyacrylamide gel. C: CsKTN1S (CCMC)-His, M: CsKTN1F (mp mutant)-His, +: present, −: absent. (b) Microtubule-shearing activity of CsKTN1 in vitro. The shearing situation of microtubules was observed by fluorescence microscopy. Bar = 10 μm.
To investigate whether the microtubule-shearing function of CsKTN1 was affected by amino substitution in mp mutant, we examined the shearing activity of CsKTN1S-His and CsKTN1F-His proteins. The taxol-stabilized and rhodamine-labeled microtubules were observed under fluorescence microscopy after perfusion with two recombinant proteins, CsKTN1S-His and CsKTN1F-His. As a result, CsKTN1S-His mediated complete microtubule shearing, while CsKTN1F-His mediated less rate of microtubule shearing in vitro (Fig. 5b). These results suggested that the amino acid substitution in the conserved domain of CsKTN1 gene may affect the microtubules binding and shearing ability of target protein.
DISCUSSION
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Plant architecture is a key factor affecting photosynthetic efficiency and cultivation pattern of crops. Optimal plant architecture enables plant to have a superior form for light capture, which could boost planting density and increase the light energy utilization during the growth and development phase, hence improving the economic output[4,36]. Miniature or semi-dwarf wheat and rice varieties have been developed and cultivated worldwide, initiating a 'green revolution' in crop breeding and production[2,5]. In this study, a novel cucumber plant architecture mutant was identified from an EMS-induced mutagenesis population, which was regulated by Katanin p60 protein. The mutant with homologs mp locus exhibited extremely miniaturized phenotypes in various organs (Fig. 1). It also possesses both female sterile and male fertile characteristics, and the resultant offspring following the cross with normal cucumber plants in producing heterozygous mp locus showed potentially useful downsizing phenotypes (Fig. 1). Thus, it could be used not only as a good resource for cucumber hybrid production, but also as a good variety resource for vine length reduction, which might provide impetus for a new direction of breeding varieties with useful downsizing plant architecture to obtain better economic benefits.
Using BSA-seq and map-based cloning methods, we identifiedand cloned the causal gene (Csa7G435510) underlying cucumber micro-plant mutant (Fig. 3). Csa7G435510 encodes a Katanin p60 protein that contains an AAA+-type ATPase domain which was highly conserved among homologous proteins from other species. Thus, it was designated as CsKTN1 (Fig. 4a, 4b). A single nucleotide substitution (C1058T) in the coding region of CsKTN1 leading to an amino acid substitution (S353F) was responsible for the plant miniatures (Fig. 4a, Supplemental Fig. S2). The mutation caused the down-regulation of CsKTN in various tissues in the mutant except for the tendril (Fig. 4c). This may be due to the feedback effect of protein inactivation on gene transcription. This phenomenon of mutation in the coding sequence of gene rather than promoter region causing changes in gene expression has been reported in several mutants of cucumber[9−10, 14].
CsKTN1 gene has been reported to control cucumber fruit length. A non-synonymous mutation of CsKTN1 resulted in short fruit3 (sf3) mutant[37]. The mutation sites of CsKTN1 gene in sf3 mutant and mp mutant are different, but the phenotypes of these two mutants are similar, such as shorter fruit, which confirmed the accuracy of CsKTN1 as a candidate gene for mp mutant. The ability of Katanin p60 protein to regulate plant morphology has great potential in the cultivation of diverse plant architecture varieties of cucumber.
Plant architectural modifications could be caused by the Katanin p60 mutation in other plants. In Arabidopsis, the Katanin p60 mutations, fra2, Bot1 and lue1, showed the micro-plant phenotype[23,24,26]. In rice, the dg1 mutant, a Katanin p60 mutation, exhibited dwarf, reduced organ size and short root phenotype[27]. In cotton, the Katanin-silenced plant showed dwarf phenotype with shorter internodes, and produced dark green and smaller leaf blades with shorter petioles[38]. Phylogenetic analysis showed that Katanin p60 proteins of cucumber had highly conserved domain and close evolutionary relationship with homolog proteins in other plants, implying that Katanin p60 homologs may share a regulatory function. However, phenotypic differences in ktn1 mutations still exist in different species. For example, in Arabidopsis and rice, ktn1 decreased fertility and produced less fertile seeds compared to wild type[26,27], while in cucumber, it resulted in female sterility with no active seeds. In addition, F1 hybrids from the crosses of ktn1 and wild type in cucumber and rice showed intermediate phenotypes (Fig. 1), indicating that mutant trait was regulated by an incomplete dominant gene[27], while in Arabidopsis, the trait was regulated by a recessive gene[24]. These discrepancies could be caused by species differences or the different mutation sites in KTN1 gene.
As the cytoskeleton, microtubules participate in a series of important life activities in cells, such as maintenance of cell structure, intracellular material transport, and mitosis[15]. The microtubules are in a state of controllable instability and dynamics, and are precisely regulated by the microtubule-associated protein (MAP) to maintain normal physiological functions of cells[39]. Several MAPs have been identified in plants and animals that are involved in promoting microtubule stabilization or destabilization[40]. The ability of Katanin p60 protein to bind and shear microtubules in vitro has also been found in Arabidopsis[16]. We confirmed that the CsKTN1 protein is a MAP by co-precipitation and microtubule shearing experiments (Fig. 5). In addition, the ability of CsKTN1 mutant protein to bind and shear microtubules decreased, indicating that the mutated amino acid has a negative impact on the function of CsKTN1 (Fig. 5). We speculate that the change of dynamic state of microtubules was the direct reason for the change of cell structure and eventually led to the change of plant architecture of the mp mutant.
In conclusion, we propose that a mutation in the CsKTN1 gene encoding Katanin p60 protein caused the changes in cucumber plant architecture. The results obtained in this paper will be useful for elucidating the mechanisms of Katanin p60 protein regulating cucumber plant architecture.
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About this article
Cite this article
Song M, Fu W, Wang Y, Cheng F, Zhang M, et al. 2022. A mutation in CsKTN1 for the Katanin p60 protein results in miniature plant in cucumber, Cucumis sativus L.. Vegetable Research 2:3 doi: 10.48130/VR-2022-0003
A mutation in CsKTN1 for the Katanin p60 protein results in miniature plant in cucumber, Cucumis sativus L.
- Received: 22 October 2021
- Accepted: 11 February 2022
- Published online: 24 February 2022
Abstract: Katanin, a microtubule shearing protein, plays an important role in plant architecture formation. However, little is known about its mechanisms in regulating plant architecture in cucumber. In the present study, through EMS mutagenesis, we identified a novel micro-plant (mp) mutant in the North China type inbred line CCMC that may be of value for cucumber breeding. The size and number of stem cells were altered in the mp mutant. Through bulked segregant analysis (BSA) sequencing approach combined with genetic mapping, the mp locus was delimited to an interval of 130.9-kb. Multiple lines of evidence suggested that the mp mutation is due to a single nucleotide polymorphism in Csa7G435510 that is predicted to encode the Katanin p60 subunit protein (CsKTN1). The expression levels of CsKTN1 decreased significantly in all tissues except the tendril of mp mutant. Subcellular localization showed that both wild-type and mutant CsKTN1 proteins were located in cell membrane, cytoplasm and nucleus of tobacco leaf cells. The mutant protein lost part of its ability to bind and shear microtubule in vitro. These findings provide new insight into the regulatory function of microtubule-shearing protein, Katanin p60, in plant architecture of cucumber.
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Key words:
- Cucumber /
- Plant architecture /
- Micro-plant /
- Katanin p60 protein /
- Microtubule shearing