1. 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 significantly 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 influence 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). Our previous study showed that the external application of MdCLE8 synthetic peptide (MdCLE8p) had no significant effect on the normal growth of Arabidopsis, showing similar activity to AtCLE3p [36,41].
2. Ectopic expression of MdCLE8 inhibits lateral root formation in Arabidopsis under nitrogen deficiency
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-deficiency 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 significantly inhibited in N-deficient 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 significantly reduced (Fig. 2B and 2C-D). The above results revealed that overexpression MdCLE8 inhibited lateral root growth and development under nitrogen deficiency condition.
3. MdCLE8 inhibits lateral root formation in apple adventitious roots under nitrogen deficiency
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 fluorescence were removed under fluorescence 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-deficiency 1/2 MS for 30 days, respectively.
Our results showed that prolonged nitrogen deficiency increased the empty expression vector transgenic adventitious roots length, but significantly decreased the lateral root tips number and density (Fig. 3A, 3C and 3E-G), this finding indicating that prolonged nitrogen deficiency 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 significantly decreased (Fig. 3A-B and 3E-G). Moreover, nitrogen deficiency intensified 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 significantly, 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 deficiency treatment.
4. 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 deficiency. 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-deficiency 1/2 MS medium for 7 days were not significantly 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 influx vector gene LAX3 (Swarup et al. 2008), cell wall remodeling gene PGAZAT, PGLR, XTR6 and EXP17 (Laskowski et al. 2006; González-Carranza et al. 2007; 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 deficiency 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 deficiency conditions. In conclusion, MdCLE8 may inhibit the formation of lateral root by affecting lateral root emergence stage.