A Peptide Encoding Gene MdCLE8 Regulates Lateral Root Development in Apple

Nitrogen is not only an essential nutrient for plant, but also an important signal molecule to integrate and regulate gene expression, metabolism and growth. Plant peptides are considered as a new hormone, and play an important regulatory role in plant growth and development. However, there are few researches on the co-regulation network between nitrogen and peptide hormones in plant. Here we identied an apple MdCLE8 gene, which encodes a putative peptide, induced by nitrogen deciency in apple. Ectopic expression of MdCLE8 inhibited lateral root formation in Arabidopsis under nitrogen deciency. Similarly, overexpression of MdCLE8 inhibited lateral root development in apple adventitious roots, and this inhibition was amplied under nitrogen deciency treatment. Further studies showed that MdCLE8 may inhibit the expression of several key genes during lateral root emergence stage in Arabidopsis, thereby inhibiting the emergence of lateral root from root cortex cells. Collectively, our study not only broadened the gene regulatory network under the inuence of nitrogen in apple, but also expanded the function of CLE peptide hormones in apple.


Introduction
Nitrogen (N) is not only one of the most important nutrients for plants, but also a signal substance to affect plant growth and development (Vidal et al. 2010(Vidal et al. , 2013. Root system architecture (RSA) plays an important role in nutrient and water absorption, and exhibits considerable developmental plasticity to changing environmental conditions (Malamy 2005;Osmont et al. 2007). Lateral root (LR) accounts for most of the total root system and displays higher plasticity to external nutrient utilization than primary root (Giehl et al. 2014). In Arabidopsis, nitrogen is an important environmental factor affecting lateral root development: Local nitrate treatment can not only stimulate the initiation of lateral root (Vidal et al. 2013), but also signi cantly promote lateral root elongation (Zhang and Forde 1998;Remans et al. 2006a); Mild nitrogen de ciency can promote the growth of lateral root, while prolonged nitrogen de ciency or excessive nitrogen supply is not conducive to lateral root growth ( In this process, the cell layer provides the greatest resistance, which may affect the number of lateral root. To reduce this resistance, cells covering the lateral root primordium undergo cell wall remodeling (CWR) to promote cell separation and thus expand the growth pathway of lateral root primordium, and a process requires proper spatiotemporal expression of cell wall remodeling genes (Lewis et al. 2013 Lewis et al. 2013). During lateral root emergence, auxin induces the expression of LAX3 in cortical cells directly covering lateral root primordium, which is also where some CWR genes are speci cally expressed. LAX3 encodes a high a nity auxin in ux carrier and functions in lateral root emergence by targeting the auxin-inducible expression of CWR genes (Swarup et al. 2008;Kumpf et al. 2013). In addition, peptide signaling also plays a key role in lateral root emergence by regulating the expression of CWR genes. are the two best characterized members, which play key roles in stem cell population maintenance in shoot apical meristem and root apical meristem, respectively (Fletcher 1999;Hobe et al. 2003). The CLE gene is also widely present in other species (Goad et al. 2017). We previously identi ed 25 MdCLE genes in the apple genome, and they have very similar structural characteristics to CLE genes reported in other species (Zhang et al. 2021), but their functions in apple are unknown.
Here, we identi ed and analyzed the function of the MdCLE8 gene, which is adjacent to CLE1/3/4/7 of Arabidopsis in the evolutionary tree and encodes highly similar CLE motif ( Fig. S1 and S2). In this study, we found that the expression of MdCLE8 was signi cantly induced by nitrogen de ciency in apple. Overexpression of MdCLE8 inhibited lateral root development in apple adventitious roots, and this inhibition was further enhanced by nitrogen de ciency treatment. Further studies showed that MdCLE8 may inhibit lateral roots formation by affecting the expression of related genes during lateral root emergence.
The Columbia ecotype (Col-0) Arabidopsis seeds were cultured on 1/2 MS medium for 4 days, then transferred to new 1/2 MS medium or N-de cient 1/2 MS medium (Nitrogen concentration was 0.1 mM, KNO 3 was replaced by KCl and nitrogen was supplemented by NH 4 NO 3 ) for further growth for 7 days.
To determine the expressions of MdCLE genes in response to different nitrogen concentrations, onemonth-old 'Pingyitiancha' (Malus × hupehensis) apple seedlings were pretreated with 1/2 medium for 3 d, then they were treated with 0 mM, 0.1 mM, 1 mM and 10 mM nitrogen medium (KNO 3 was replaced by KCl and nitrogen was supplemented by NH 4 NO 3 ) for the indicated times, respectively. The seedlings were growning at 24 C with a 16 h light/8 h dark photoperiod.

Vector construction and plant transformation
The open reading frame (ORF) of MdCLE8 was fused to pRI-GFP to generate 35S::MdCLE8-GFP. Then 35S::MdCLE8-GFP and empty expression vector were introduced into Agrobacterium rhizogenes strain K599 and transformed into 'M26' apple stem segments to induce hairy roots (Zhou et al. 2019), and the empty expression vector (CK) was used as a control. GFP uorescence of transgenic adventitious roots was identi ed by uorescence microscopy.

Gene expression analysis
Total RNA was extracted from apple seedlings, 'M26' transgenic adventitious roots, and Arabidopsis using RNA Plant Plus reagent (Tiangen, Beijing, China) according to the instructions, and reverse transcription assay was performed by using the PrimeScript cDNA Synthesis Kit (Takara, Liaoning, China). RT-qPCR was performed with the UltraSYBR mixture (Takara, Liaoning, China) by an ABI7500 RT-PCR system ). Primers used for RT-qPCR are listed in supplementary Table S1, and 18S rRNA was used as an internal control.

GUS staining
After germinating and growing for 4 days on 1/2 MS medium, DR5-GUS and DR5-GUS/MdCLE8-OE transgenic Arabidopsis seeds were transferred to N-de cient 1/2 MS medium for 3 days. 7-day-old transgenic Arabidopsis seedlings were stained using a GUS solution (Clough and Bent 1998), then the total number of lateral root primordium was counted under the type microscope.

Determination of polygalacturonase and pectinase activities
Polygalacturonase and pectinase activity detection kits were purchased from Comin Biotechnology (Suzhou, China).

Root measurement
For Arabidopsis, the primary root length and the lateral root number were analyzed by Digimizer software. For apple seedlings, the phenotypes of apple transgenic adventitious roots were scanned with a root scanner (Perfection V850 Pro Photo, Epson), and the lateral root length and number were measured with Digimizer software. 2.7. Statistical analysis SPSS v17.0 software was used for statistical analysis. Statistical analysis was performed using a Student's t-test, where ns P > 0.05, *P < 0.05 and **P < 0.01. Different letters indicate signi cant difference (P < 0.05) as obtained by one-way ANOVA test.  Table S1.

MdCLE8 is a nitrogen-responsive gene that encodes a putative peptide in apple
Nitrogen is one of the essential elements for plant growth and development. In order to investigate the relationship between nitrogen and MdCLE peptide hormones in apple, we quantitatively detected the expression levels of MdCLE genes in subfamily III under different nitrogen concentrations (Fig. S1). With the decrease of nitrogen concentration in nutrient solution, the expression levels of MdCLE8 and MdCLE23 in apple seedling were signi cantly induced, while the other detected that MdCLEs remained basically unchanged (Fig. 1). These results suggested that MdCLE8 and MdCLE23 might be functional genes under the in uence of nitrogen in apple.
To investigate further, we cloned the MdCLE8 gene. By sequence alignment, we found that MdCLE8 protein had a conserved CLE motif at the C terminal similar to that of AtCLE1/3/4/7 (Fig S2) To investigate the function of MdCLE8 gene, we constructed MdCLE8 overexpression vector, and transformed it into wild type Arabidopsis (MdCLE8-OE1, MdCLE8-OE2, and MdCLE8-OE3). Subsequently, we treated transgenic Arabidopsis lines with 1/2 MS and N-de ciency 1/2 MS medium, and used the wild type (WT) as a control. The results showed that there was no difference between WT and MdCLE8-OE transgenic Arabidopsis growing on 1/2 MS medium, and the primary root length, lateral root number and density statistics all indicate this ( Fig. 2A and 2C-D). In contrast, the growth of MdCLE8-OE transgenic Arabidopsis was signi cantly inhibited in N-de cient 1/2 MS medium. Compared with the WT, the primary root length of MdCLE8-OE transgenic Arabidopsis was slightly reduced, and the lateral root number and density were signi cantly reduced ( Fig. 2B and 2C-D). The above results revealed that overexpression MdCLE8 inhibited lateral root growth and development under nitrogen de ciency condition.

MdCLE8 inhibits lateral root formation in apple adventitious roots under nitrogen de ciency
To explore the function of MdCLE8 on the growth of apple root system, the 35::MdCLE8-GFP vector and empty expression vector pRI-GFP (CK) were transformed into 'M26' apple shoot base cells by Agrobacterium rhizogenes-mediated genetic transformation (Fig. S3A). The false positive adventitious roots without GFP uorescence were removed under uorescence microscope (Fig. S3B), and apple seedlings were transplanted into vermiculite without any nutrition. Finally, the apple seedlings were watered with 1/2 MS or N-de ciency 1/2 MS for 30 days, respectively.
Our results showed that prolonged nitrogen de ciency increased the empty expression vector transgenic adventitious roots length, but signi cantly decreased the lateral root tips number and density (Fig. 3A, 3C and 3E-G), this nding indicating that prolonged nitrogen de ciency will seriously affect the growth and development of apple lateral root. Under the 1/2 MS condition, the total lateral root tips number and density of 35S::MdCLE8-GFP transgenic adventitious roots were signi cantly decreased (Fig. 3A-B and 3E-G). Moreover, nitrogen de ciency intensi ed the effect of 35S::MdCLE8-GFP on the growth of apple adventitious roots (Fig. 3C-G). The root length of MdCLE8-OE transgenic adventitious roots decreased signi cantly, the lateral root number decreased from 45.64-79.58%, and the lateral root density decreased from 44.37-68.71%. Taken together, our results indicate that MdCLE8 inhibited lateral root formation in apple, and this inhibition was enhanced by nitrogen de ciency treatment.

MdCLE8 is involved spatiotemporal regulation of key genes during lateral root emergence
The above experimental results revealed that the overexpression of MdCLE8 gene inhibited lateral root formation in Arabidopsis under nitrogen de ciency. An unsolved question is what the underlying mechanism is. To answer this question, we obtained a hybrid Arabidopsis material of DR5-GUS and MdCLE8-OE (DR5-GUS/MdCLE8-OE). The development of lateral root primordia of DR5-GU5/MdCLE8-OE was observed by GUS staining (Fig. S4A), and DR5-GUS was used as control. The results showed that the total lateral root primordia number and density of DR5-GUS/MdCLE8-OE on N-de ciency 1/2 MS medium for 7 days were not signi cantly different from control (Fig. S4B-D), suggesting that MdCLE8 does not affect lateral root primordia formation in Arabidopsis.
We hypothesized that the inhibition of MdCLE8-OE on lateral root development was caused by affecting lateral root emergence stage. Therefore, we examined the expression levels of key genes in Arabidopsis that affect the lateral root emergence, including auxin in ux vector gene LAX3 (Swarup et al. 2008 Kumpf et al. 2013). The results showed that overexpression of MdCLE8 inhibited the expression of LAX3, PGAZAT, PGLR and XTR6 genes in Arabidopsis under nitrogen de ciency conditions (Fig. 4A). At the same time, we detected the expression levels of MdLAX3-like and MdXTH genes, as well as polygalacturonase (PG) and pectinase activity in apple. The results showed that overexpression of MdCLE8 inhibited the expression of some MdLAX-like and MdXTH genes (Fig. 4B-C), and decreased the activities of PG and pectinase (Fig. 4D-E), and this inhibition was particularly obvious under nitrogen de ciency conditions. In conclusion, MdCLE8 may inhibit the formation of lateral root by affecting lateral root emergence stage. Our previous research has shown that 1 µM MdCLE8p had no signi cant effect on the root system of Arabidopsis cultured in 1/2 MS medium (Zhang et al.). Similarly, in this study, we found that the ectopic overexpression of MdCLE8 did not affect the growth of Arabidopsis in 1/2 MS medium, but signi cantly inhibited lateral root formation in N-de ciency 1/2 MS medium (Fig. 2). Furthermore, we also overexpressed the MdCLE8 gene in apple adventitious roots. We found that MdCLE8 gene also inhibited apple lateral roots formation, and this inhibition was further enhanced by nitrogen de ciency treatment (Fig. 3), suggesting that MdCLE8 gene and nitrogen de ciency signal co-regulate the development of apple lateral roots.

Discussions
Lateral root formation in plants is a complex physiological process. To put it simply, lateral root primordia are rst produced from xylem pole pericycle cells, and then further developed and grow until they successfully drill out of the parent root epidermis to produce lateral roots (Malamy and Benfey 1997;Casimiro et al. 2001;Dubrovsky et al. 2001). The growth of the lateral root primordium needs to pass through three cell layers, and the substances in the cell layer such as pectin provide resistance to this process (Lewis et al. 2013). Our results further showed that the overexpression of MdCLE8 strongly inhibited the expression of cell wall remodeling genes (Fig. 4), thus impeding the normal separation of the cell layer. Therefore, we suggest that the in uence of MdCLE8 on lateral root formation may be due to the growth and emergence of lateral root primordium rather than the origin of lateral root primordium.
Previous studies have shown that the CLE-CLV1 signaling pathway is a core module that regulates the expansion of the lateral root system of Arabidopsis under low nitrogen conditions (Araya et al. 2014b, a). The MdCLE8 protein in apple has a highly similar CLE motif to that of AtCLE1/3/4/7 in Arabidopsis ( Fig  S2), and MdCLE8 and AtCLE3 have similar regulatory functions for plant lateral root formation (Fig. 2), suggesting that CLE genes with similar CLE motif in different species are functionally conserved. However, more detailed anatomical analysis of apple root system, and the receptors that CLE peptides act on remain to be studied. Therefore, the speci c mechanism by which MdCLE8 regulates lateral root development in apple still needs to be further analyzed.

Declarations
Data availability statement All data generated or analysed during this study are included in this published article [and its supplementary information les].

Declaration of competing interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.