Cunninghamia lanceolata PSK Peptide Hormone Genes Promote Primary Root Growth and Adventitious Root Formation

Phytosulfokine-α (PSK-α) is a newly discovered short peptide that acts as a phytohormone in various plants. Previous studies have shown that PSK-α is critical for many biological processes in plants, such as cell division and differentiation, somatic embryogenesis, pollen germination and plant resistance. In this study, we cloned two PSK homolog genes from Cunninghamia lanceolata (Lamb.) Hook (Chinese fir), ClPSK1 and ClPSK2, and characterized their function in root development. Quantitative RT-PCR analyses showed that both ClPSK1 and ClPSK2 were expressed in vegetative organs, mainly in roots. Transgenic Arabidopsis plants overexpressing ClPSK1 or ClPSK2 showed a higher frequency of adventitious root formation and increased root length. The expression of genes in Arabidopsis that are involved in stem cell activity (PLT1, PLT2 and WOX5), radial organization of the root (SHR and SCR) and cell cycle (CYCB1;1, CYCD4;1, CDKB1;1 and RBR) were significantly up-regulated, which may contribute to the elongation of the primary root and the formation of adventitious root in transgenic lines. Our results suggest that ClPSKs play an important role during root growth and development.


Introduction
Cunninghamia lanceolata (Lamb.) Hook (Chinese fir) is an important native evergreen tree in southern China. Because of its elite wood attributes and high timber productivity, Chinese fir occupies a prominent role regarding ecological and commercial prospects.
In natural environments, the growth and development of plants are strongly influenced by surrounding conditions. This plasticity allows plants to adapt to a changing and adverse environment, thus compensating for their immobile lifestyle. In many cases, the plant's response to developmental conditions is mediated by non-proteinaceous phytohormones, such as auxin, cytokinin, gibberellin, abscisic acid, ethylene, etc.
3 species transgenesis studies in Arabidopsis thaliana, we found that the rate and frequency of adventitious root formation was increased by over-expression of ClPSK corresponding to an increase in the expression levels of PLT1, PLT2, SHR, SCR and WOX5. These results indicate that ClPSK may play an important role in adventitious root formation. We demonstrate that overexpression of ClPSK improves root growth and promotes formation of adventitious root in Arabidopsis thaliana. These findings may aid future studies aimed at improving Chinese fir adaptability.

Molecular Cloning and Characterization of the ClPSK Genes
We cloned the ClPSK1 and ClPSK2 sequences from Chinese fir embryogenic callus. The cDNA of ClPSK1 is 303 bp long and the open reading frame encodes 100 amino acids. The cDNA of ClPSK2 is 294 bp long, encoding 97 amino acids. PSK has a predicted secretion signal at the N-terminus and a highly conserved single PSK domain (YIYTQ) close to the C-terminus (Figure 1a). Sequence alignment reveals that the PSK domain is identical to that of other known PSK precursor amino acid sequences ( Figure 1a). However, the remaining sequence is highly divergent across species. Relative expression levels of ClPSK1/2 in the root, stem, leaf and embryonic callus of Chinese fir as determined by quantitative real-time PCR analysis. Error bars represent the standard deviations of three independent biological replicates. Different letters indicate significant differences between treatments, significant differences in mRNA levels were detected by the ANOVA test (P < 0.05).
To investigate the spatial distribution of ClPSK transcripts, quantitative real-time PCR (qRT-PCR) analysis was performed in different tissues of Chinese fir. This analysis indicated that ClPSK was expressed in the root, stem, leaf and embryonic callus of Chinese fir, and that the relative expression of ClPSK1 and ClPSK2 genes was highest in roots (( Figure 1b).
To investigate the spatial distribution of ClPSK transcripts, quantitative real-time PCR (qRT-PCR) analysis was performed in different tissues of Chinese fir. This analysis indicated that ClPSK was expressed in the root, stem, leaf and embryonic callus of Chinese fir, and that the relative expression of ClPSK1 and ClPSK2 genes was highest in roots (( Figure 1b).

Overexpression of ClPSK Promotes Root Growth in Arabidopsis Thaliana
To determine functionality of ClPSK1 and ClPSK2, we overexpressed ClPSK genes under the control of the CaMV 35S promoter in Arabidopsis thaliana. We found that the roots of transgenic plants were significantly longer than those of the wild type ( Figure 2a). This effect was consistent across three independent transgenic lines for each gene, with root lengths increased by 39.4%, 50.2% and 38.5% in the transgenic lines ClPSK1-1, ClPSK1-2 and ClPSK1-3, respectively, and increased by 23-28% in lines ClPSK2-1, ClPSK2-2 and ClPSK2-3, compared to the wild type ( Figure 2b). These results demonstrate that overexpressing ClPSK in Arabidopsis thaliana promotes primary root growth.

Overexpression of ClPSK Gene Promotes Organ Differentiation
Previous studies have shown that PSK-α can promote organogenesis in plants [21]. To explore the function of ClPSK, we observed organogenesis from 35S::ClPSK and wild-type callus treated with basic liquid Murashige and Skoog (MS) medium. We found no apparent changes in wild-type callus after liquid suspension culture for 7 days, while transparent protrusions could be observed on the surface of 35S::ClPSK callus (Figure 3a-l). In addition, we observed transparent protrusions from wild-type callus grown in basic liquid MS medium supplemented with 0.1 mg L −1 PSK-α after 7 days of liquid culture (Figure 3a-l). After 14 days, the transparent protrusions became significantly longer ( Figure 3m). Observed through the microscope, we found the transparent protrusions formed on the callus surface to display obvious root characteristics (Figure 3n-p). These results demonstrate that ClPSK can induce organogenesis and lead to the production of adventitious roots on callus surface in Arabidopsis thaliana. The center line represents the mean, and error bars represent standard error (SE). n ≥ 20 biological replicates. Asterisks indicate statistically significant difference between WT and transgenic lines, as determined by Student's t-test (P < 0.05).

Overexpression of ClPSK Gene Promotes Organ Differentiation
Previous studies have shown that PSK-α can promote organogenesis in plants [21]. To explore the function of ClPSK, we observed organogenesis from 35S::ClPSK and wild-type callus treated with basic liquid Murashige and Skoog (MS) medium. We found no apparent changes in wild-type callus after liquid suspension culture for 7 days, while transparent protrusions could be observed on the surface of 35S::ClPSK callus (Figure 3a

Overexpression of ClPSK Genes Upregulates the Expression of Genes Related to Root Morphogenesis
To further investigate the relationship between ClPSK1/2 and root formation, we analyzed the expression of genes associated with root development, such as PLT1, PLT2, SHR, SCR and WOX5 in Arabidopsis callus. We found that these genes were up-regulated in callus overexpressing ClPSK genes or treated with PSK-α cultured for 7 days in suspension culture, with ClPSK overexpression being more effective at inducing some of these genes compared to PSK-α treatment (Figure 4a). These data support at a molecular level that overexpression of ClPSK promotes root morphogenesis.

Overexpression of ClPSK Genes Upregulates the Expression of Genes Related to Root Morphogenesis
To further investigate the relationship between ClPSK1/2 and root formation, we analyzed the expression of genes associated with root development, such as PLT1, PLT2, SHR, SCR and WOX5 in Arabidopsis callus. We found that these genes were up-regulated in callus overexpressing ClPSK genes or treated with PSK-α cultured for 7 days in suspension culture, with ClPSK overexpression being more effective at inducing some of these genes compared to PSK-α treatment (Figure 4a). These data support at a molecular level that overexpression of ClPSK promotes root morphogenesis.  Different letters indicate statistically significant differences between treatments in one genotype. Relative expression data are plotted as the mean ± SD, n = 3 biological replicates, and statistically significant differences from WT (P < 0.05) were obtained using an ANOVA test.
Root meristem activity is required to accelerate the rate of cell division for a continuous supply of new cells. Acceleration of cell elongation contributes to enhanced root growth, but it is not sufficient to promote long-term growth without the production of new cells from the meristem. Therefore, we tested the expression level of genes involved in cell division [25][26][27][28][29][30]. The expression levels of CYCB1;1, CYCD4;1, CDKB1;1 and RBR are up-regulated in transgenic lines and wild-type treated with 0.1mg L −1 PSK-α, which were measured after callus was cultured for 7 days in suspension culture (Figure 4b). Moreover, the expression levels of CDKB1;1 and RBR of transgenic lines were slightly higher than PSK-α-treated wild-type (Figure 4b). These results indicate that PSK is able to promote cell division.

Discussion
Here, we describe the molecular cloning and characterization of two ClPSK gene homologs originating from Chinese fir, that encode the precursor of PSK-α. We detected high expression of both ClPSK1 and ClPSK2 genes in Chinese fir root (Figure 1b). Previous studies have shown that the primary root of plants overexpressing AtPSK4 is significantly longer than in the wild type by promoting root growth by enhancing cell elongation [9,40], and that GhPSK can promote the elongation of roots in Arabidopsis thaliana [41]. In this study, our results show that overexpression of ClPSK1 or ClPSK2 can significantly promote root growth in Arabidopsis thaliana, consistent with the phenotypes of AtPSK4 and GhPSK overexpression in Arabidopsis thaliana.
The growth and development of plant roots involves complex regulatory networks, as well as key transcription factors, such as PLT1, PLT2, SHR, SCR and WOX5, which have been shown to be involved in root formation. We observed that ClPSK1/2 overexpression or PSK-α treatment induced adventitious roots on the callus surface after 14 days. Correlating with these findings, increased levels of ClPSK1/2 enhanced the expression levels of PLT1, PLT2, SHR, SCR and WOX5 (Figure 4a). Taken together, these data suggest that ClPSK1 and ClPSK2 effect gene networks related to root Different letters indicate statistically significant differences between treatments in one genotype. Relative expression data are expressed as the mean ± SD, n = 3 biological replicates, and statistically significant differences from WT (P < 0.05) were obtained using an ANOVA test. (b) ClPSK1/2 induce expression of cell cycle genes in Arabidopsis callus. Relative expression levels of cell-cycle genes in wild-type (WT), PSK-α treated (PSK-α) and 35S::ClPSK1/2 (ClPSK1 and ClPSK2) callus grown 7 days in basic MS liquid medium. Both ClPSK1/2 overexpression and exogenous treatment upregulate expression of RBR, CDKB1;1, CYCB1;1 and CYCD4;1. Different letters indicate statistically significant differences between treatments in one genotype. Relative expression data are plotted as the mean ± SD, n = 3 biological replicates, and statistically significant differences from WT (P < 0.05) were obtained using an ANOVA test.
Root meristem activity is required to accelerate the rate of cell division for a continuous supply of new cells. Acceleration of cell elongation contributes to enhanced root growth, but it is not sufficient to promote long-term growth without the production of new cells from the meristem. Therefore, we tested the expression level of genes involved in cell division [25][26][27][28][29][30]. The expression levels of CYCB1;1, CYCD4;1, CDKB1;1 and RBR are up-regulated in transgenic lines and wild-type treated with 0.1mg L −1 PSK-α, which were measured after callus was cultured for 7 days in suspension culture ( Figure 4b). Moreover, the expression levels of CDKB1;1 and RBR of transgenic lines were slightly higher than PSK-α-treated wild-type (Figure 4b). These results indicate that PSK is able to promote cell division.

Discussion
Here, we describe the molecular cloning and characterization of two ClPSK gene homologs originating from Chinese fir, that encode the precursor of PSK-α. We detected high expression of both ClPSK1 and ClPSK2 genes in Chinese fir root (Figure 1b). Previous studies have shown that the primary root of plants overexpressing AtPSK4 is significantly longer than in the wild type by promoting root growth by enhancing cell elongation [9,40], and that GhPSK can promote the elongation of roots in Arabidopsis thaliana [41]. In this study, our results show that overexpression of ClPSK1 or ClPSK2 can significantly promote root growth in Arabidopsis thaliana, consistent with the phenotypes of AtPSK4 and GhPSK overexpression in Arabidopsis thaliana.
The growth and development of plant roots involves complex regulatory networks, as well as key transcription factors, such as PLT1, PLT2, SHR, SCR and WOX5, which have been shown to be involved in root formation. We observed that ClPSK1/2 overexpression or PSK-α treatment induced adventitious roots on the callus surface after 14 days. Correlating with these findings, increased levels of ClPSK1/2 enhanced the expression levels of PLT1, PLT2, SHR, SCR and WOX5 (Figure 4a). Taken together, these data suggest that ClPSK1 and ClPSK2 effect gene networks related to root development, and that they have functions involved in root morphogenesis. The positive contribution of PSK signaling to promoting adventitious root formation from callus has not been reported previously, although PSK has been implicated in inducing adventitious root formation from cucumber hypocotyls and adventitious bud formation from callus of Antirrhinum majus [21,42].
In addition, we found that expression of the cell cycle marker genes (CYCB1;1, CYCD4;1, CDKB1;1 and RBR) was up-regulated in the adventitious roots of 35S::ClPSK1/2 transgenic lines (Figure 4b). CYCD4;1 is abundantly expressed during the initiation of the root primordium, and CYCD4;1 transcripts accumulate in the vascular tissue of roots, as well as in lateral root primordia [29]. Overexpression of CYCB1;1 can promote root meristem division and increases root elongation [35]. CDKB is a type of CDK kinase that acts in the S and G2/M phases of the cell cycle [43,44]. Inhibition of CDKB activity can cause serious defects in plant meristems [45]. The expression of RBR directly affects the number and characteristics of cells in the root apical meristem (RAM) in Arabidopsis thaliana. Furthermore, RBR is required for stem cell maintenance, cell differentiation and lateral organ production [46]. These genes are all involved in the cell-cycle, but how does PSK impinge on the molecular control of the cell cycle? Hormone signaling is a key component of root growth, as a plant's response to changing environmental or developmental conditions are mediated by hormones. PSK is a new plant hormone, but its physiological characteristics and its mechanism of action are still unclear. Our study demonstrates that ClPSK gene promotes organ differentiation, and it highlights a novel plant growth regulator that may be used for organogenesis and somatic embryogenesis of Chinese fir. Our findings provide a preliminary understanding of ClPSK function, and give an important basis for further understanding the PSK signal transduction mechanism and its role in plant growth and development.

Plant Materials and Culture Conditions
Experiments were performed with Arabidopsis thaliana ecotype Columbia (Col-0). Arabidopsis seeds were surface-sterilized with 75% (v/v) ethanol for 30 s, treated with 0.1% (w/v) HgCl 2 for 2.5 min, germinated on Murashige and Skoog (MS) medium, and cultured at 22°C and 70% humidity with a 16/8 h light/dark cycle. For the measurement of root length of T3 homozygous plants were measured after growing for 15 day after germination.
For callus induction, Arabidopsis thaliana seeds germinated on MS medium for 2 weeks, then Arabidopsis leaves were transferred to MS solid medium containing 2 mg L −1 2,4-dichlorophenoxyacetic acid, 0.2 mg L −1 , benzyl-aminopurine, 500 mg L −1 casein hydrolysate (CH) (Sigma, Darmstadt, Germany) and 30 g L −1 sucrose, after which they were cultured at 23°C in darkness. Callus was transferred to basic MS liquid medium consisting 30 g L −1 sucrose, and cultured at 23°C in darkness so as to induce the formation of adventitious roots.
Roots, stems and leaves of Chinese fir seedlings germinated from somatic embryos and embryogenic callus were induced following the methods previously described by Zhou Xiaohong [47].

Gene Cloning and Construction of Vectors and ClPSK Overexpression Lines
Based on transcriptome data (shared in the Lab) of Chinese fir, the full-length cDNA clones of two ClPSK genes, ClPSK1 and ClPSK2, were cloned from embryogenic callus of Chinese fir and used the NCBI ORF finder to detect open reading frames (https://www.ncbi.nlm.nih.gov/orffinder/). Briefly, ClPSK1 and ClPSK2, were cloned and inserted into the pBI121 vector with restriction enzymes Bam HI and Xba I (NEB) to generate the overexpression vector 35S::PSK. Arabidopsis thaliana plants were transformed via the floral dipping method using Agrobacterium tumefaciens [48]. Resistant plants were selected using 50 mg L −1 kanamycin. The transgenic plants were selected by the PCR analysis. The single-locus homozygous transgenic lines were then identified by the genetic analyses of segregation at 3:1 in the T1 generation and no separations in the T2 and T3 generations (n > 30). All primers used for gene cloning were listed in Table S1.

Characterization of the ClPSK Genes Analysis
The amino acid multiple sequence alignment between ClPSK and phytosulfokine-a (PSKs) from other species was performed using TEXshade (https://ctan.org/pkg/texshade). The PSK protein sequences of other species were downloaded from the National Center for Biotechnology Information (NCBI) web site (http://www.ncbi.nlm.nih.gov).

Quantitative Real-Time PCR
Total RNA was isolated using the RNAprep Pure Plant Kit (Tiangen, Beijing, China) and then was reversely transcribed to cDNA with a reverse transcriptase kit (Roche, Shanghai, China). Quantitative real-time PCR was performed using the LightCycler 480 System (Roche Applied Science, Shanghai, China), as previously described [47]. The CleIF-3 housekeeping gene was selected as the endogenous reference gene for the qRT-PCR analysis of cambial development in Chinese fir [49], and primers for qRT-PCR are listed in the Table S2. All primers used for qRT-PCR of Arabidopsis thaliana were listed in Table S3. Each measurement was performed using three biological samples and each test of sample was conducted with three replicates. Relative gene expression was performed using the 2 −∆∆CT method [50].

Morphological Analysis
The morphology and characteristics of adventitious roots was evaluated using a stereoscope (Leica, S8AP0) and micrographs were obtained using an inverted microscope (Leica, DMI4000, Wetzlar, Germany).