Phenotype variations of grapevine cv. Muscat Hamburg after Root restriction cultivation
The shoot length of one-year-old self-rooted seedlings of Vitis vinifera L. cv. Muscat Hamburg were measured from 60 to 125 days after planting (DAP) under two different cultivations. The results showed that the new shoot length both reached to 2 meters in 60 DAP. Root restriction (referred to RR) cultivation had a rapid growth and longer than control (non-restriction, referred to nR) cultivation on 100 DAP. From 100 to 125 DAP, the shoots of nR cultivation grew faster and reached about 5.5 meters, one meter longer than that of RR cultivation (Figure S1A). The base diameters of the new shoots in nR cultivation was always higher than that of the RR cultivation (Figure S1B). Since the secondary shoots were trimmed every 7–10 days, the growth of secondary shoots was measured between two pruning times. The results showed that the number of secondary shoots in nR cultivation was 18–20 per tree, which was almost 2 folds to RR’s 8–11 per tree (Figure S1E). According to the length of the secondary shoots, RR and nR cultivations both were divided into four grades, denoted as I-IV (Figure S1C, D). It was found that the length of the secondary shoots in nR cultivation was significantly higher than that of the RR cultivation, and the length difference from grades I-IV became larger and larger. Moreover, nR cultivation was almost twice as longer as the RR cultivationin grade IV (Figure S1F). The diameter of the secondary shoots was not significantly different in grade I, but nR cultivation was significantly higher than that of RR cultivationin grade II-IV (Figure S1G).
A total of 12 comparative phenotype photographs of the root system were taken every 10 or 15 days from April 24 to August 18, and the nR and RR cultivations were recorded as nR1-12 (Figure S2) and RR1-12 (Figure S3). Two cultivations showed a similar root formation orders including absorbing roots, secondary lateral roots and new adventitious roots. Finally, the old roots degenerate and new adventitious roots developed into the main root system. However, after the 7th sampling (70 DAP), the root morphology of the two cultivations changed significantly. The main manifestations were as follows:
(1) Compared whit the nR cultivation, a large number of new adventitious roots with thinner diameters emerged in RR cultivation. Adventitious roots occurred in clusters in both cultivations (Fig. 1A, B). The number and diameter of adventitious roots in the cluster were analyzed at the 7th sampling. The results showed that the number increased significantly after RR cultivation, which was 8 per cluster and about twice than that of 4 per cluster in the nR cultivation (Fig. 1C). While the diameter of adventitious roots in RR cultivation was about 0.15 cm, which was significantly lower than the nR (about 0.22 cm) (Fig. 1D). (2) The number of lateral roots increased after RR cultivation. Comparing the roots morphology of the same position in the two cultivations of the 12th sampling (125 DAP), the lateral roots mainly distributed in the upper part of roots in nR cultivation (Fig. 1E), but in RR cultivation lateral roots densely distributed on the whole roots (Fig. 1F). (3) Growth defect ofroot tips led to a large number of clustered roots emerged inRR cultivation. Clustered roots emerged from the degenerated root tips (Fig. 1G, I), and compared with the nR cultivation, almost all root tips were degraded after the RR cultivation. The secondary roots and tertiary lateral roots were appearing (Fig. 1H, J); (4) Root regeneration was accelerated after RR cultivation. Along with the lower new lateral roots occurred, the upper lateral roots became brown and disappeared, and the overall browning rate was faster than nR cultivation (Figure S4).
Sequencing Statistics In Different Grapevine Samples
The continuous phenotypic observation of grapevine root system revealed that significant differences were occurred from the 7th to 12th samplings in RR and nR cultivations. Then the 7th and 12th sampling points were selected for small RNA sequencing. These four root samples were named as nR7, nR12, RR7, and RR12, and each sample had three replicates, recorded as A, B, and C, respectively. A total of 214,439,588 raw reads were obtained and finally got 168,741,687 clean reads after the quality control steps. The clean reads of each library were between 11.29 and 15.60 M (Table 1). The copy number of clean reads uniq ranged from one to ten were more than 96.5%, among them, single copy and two copies accounted for 69.72% and 15.24%, respectively, totally reached 85% (Figure S5).
Identification Of Known And Novel Grapevine miRNAs
After a series of miRNA prediction analysis, a total of 153 grapevine known miRNAs, and 119 novel miRNAs (named by chromosome random number) were obtained. The length distribution results showed that the known miRNAs were distributed between 19 and 24 nt (nucleotide), of which more than 60% were 21 nt miRNAs (Fig. 2A). The length of novel miRNA ranges from 18 to 25 nt, of which the first peak was 23 nt, which accounts for more than 35%, and the proportion was slightly higher in RR cultivation; the second length peak is 21 nt, which accounts for about 25% (Fig. 2B). The miRBase database recorded a total of 48 known miRNA families in grapevine, and 45 of them were detected in this study except miR828, miR2950, and miR3628 families. And only 30 miRNA members from 13 known grapevine miRNA families were not detected. The predicted novel miRNAs were used to blast miRVIT database, and 18 of them was perfected matched. Among them, nine were similar to known miRNA families in grapevine, including Un_39994, 6_13658, 19_26046, 19_26048 and 19_25033 were similar to vvi-miR477a, 1_21167 were similar to vvi-miR482, 14_36566 and 17_1792 were similar to vvi-miR3627-5p, and 14_37516 were similar to vvi-miR3633b-3p,respectively. (Table S1).
To obtain the detail in formation of the predicted grape novel miRNA, they were aligned with mature miRNAs database of miRBase by default parameters.14 conserved grapevine miRNA family members were obtained (Fig. 3). Seven of the14 conserved miRNA also detected in the miRVIT database, but that was much better matched in miRBase than miRVIT database. Among them, five novel miRNAs annotated in miRVIT that similar to vvi-miR477a matched better in miRBase database to ppt-miR477f (Physcomitrella patens).Meanwhile, 1_21167 and 14_37516 matched better with mtr-miR482-3p (Medicago truncatula) and gma-miR482a -3p (Glycine max), respectively. The remaining seven novel miRNA were6_12672, 17_2431,11_7793, 9_19848, 15_8904, 2_4979, and 9_20339, which were similar to csi-miR159b-5p (Citrus sinensis), mes-miR159a-5p (Manihot esculenta), vvi-miR396b, csi-miR156f-3p, ath-miR162a-3p (Arabidopsis thaliana), seu-miR319 (Salicornia europaea), and osa-miR396e- 3p (Oryza sativa), respectively (Table 2).In addition, there were 82 novel miRNAs aligned to miRNA families that had been reported in other species but not in grapevine (Table S3). 5_32700 and ghr-miR827a (Gossypium hirsutum), 14_36566 and vca-miR391-5p (Vrieseacarinata), 14_37655 and mes-miR1446 had an alignment score of more than 90 points, and mature sequences had high sequence homology with other species (Table S2). The remaining 23 novel miRNAs that did not match either databases were considered as grapevine-specific novel miRNAs (Table S3).
Differentially Expressed miRNAs (DEMs) Analysis
Principal component analysis (PCA) showed that the distribution was relatively concentrated among biological replicates. Moreover, nR7 and RR7 samples were close and some replicates could not be completely separated, and nR12 and RR12 could be completely separated (Figure S6). There were 26, 33, 26 and 32 differentially expressed miRNAs (DEMs) were identified in different cultivations (RR7 vs nR7; RR12 vs nR12) and different cultivation stages (nR12 vs nR7; RR12 vs RR7). Among them, both the know miRNA and novel miRNA showed up ordown regulation (Table S4). Hierarchical clustering heat map analysis showedthat different replicates of the same sample clustered together. In different cultivations, both vvi-miR3627-3p and 11_random_23 were up-regulated, while vvi-miR166a, vvi-miR482, vvi-miR2111-5p, Un_39994*, and 19_26046 showeda down-regulated expression (Fig. 4A). In different cultivation stages, 5_32700* and 18_33385 were up-regulated, and the down-regulated expressions miRNAs were vvi-miR3633a-3p, 17_2431*, and 2_4979 (Fig. 4B). In addition, in the later development stage of the nR cultivation (nR12 vs nR7), vvi-miR398a, vvi-miR3623-3p, and miR3634-3p were down-regulated, and vvi-miR167b, vvi-miR319g, 6_13658, and 14_37516 were up-regulated, which showed an opposite expression trend in RR cultivation (RR12 vs nR12) (Fig. 4B)
MiRNA expression levels analysis found that know miRNA were in higher TPM (transcript per million) values. 17 of know miRNA had TPM values higher than 30 and the highest reached to 3000 (Fig. 5A). TPM values above 10 in novel miRNA were only 11 (Fig. 5B). The highest five TPM of know miRNAs were vvi-miR3634-3p, vvi-miR166c, vvi-miR159c, vvi-miR482, and vvi-miR398b, and of novel miRNAs were 1_21167, 1_21167*, 14_37516, Un_39994*, and 11_7793.
Analysis Of vvi-miRNA Mediated Grapevine Root Formation
The number of predicted target genes in DEMs was 344, 738, 402, and 486 in different cultivations (RR7 vs nR7; RR12 vs nR12) and different cultivation stages (nR12 vs nR7; RR12 vs RR7), respectively. GO annotation analysis revealed that the predicted target genes participated in a variety of biological processes, and both included regulation of transcription, oxidation-reduction process, serine family amino acid metabolic process and defense response. In different cultivation models, the target genes were predicted function on lignin catabolic process, electron transport, and response to water deprivation (Fig. 6A). In addition, response to abscisic acid stimulus, response to salt stress, regulation of meristem growth and polarity specification of adaxial/abaxial axis were in top 10 ranks in different cultivation stages (Fig. 6B). The firstcategory in cellular components classification was the nucleus, while the protein binding and ATP binding categories were the most abundant categories in molecular functions classification. Gene function annotation found a total of 24 target genes related to root development, which corresponding to 17 vvi-miRNAs. Target genes of vvi-miR156, vvi-miR166, vvi-miR2111-5p, and vvi-miR3624-3p participated in root hair development, as well as, vvi-miR164 and vvi-miR482 affected lateral root and root cap development; target genes of vvi-miR396 annotated in root development. In target genes of novel miRNAs were functioned on more different root developments, such as the target genes of 4_24249 and 17_2431 affected primary root development while 15_8868 and 15_8867 participated in root morphogenesis. KEGG metabolic pathways analysis was conducted and some target genes had corresponding metabolic pathway annotations. Among them, miR2111-5p participated in vasopressin-regulated water reabsorption, corresponding to its GO annotation in root hair development (Table 3).
Vvi-miR160 Family Contributes To Grapevine Root Development
Root tip degradation was one of the most obvious root phenotypes after RR cultivation. MiR160 had been reported to play an important role in root tip development[28]. Five members of the vvi-miR160 family named vvi-miR160a, b, c, d and e were obtained by miRbase search. Among them, the mature sequence lengths of vvi-miR160a and vvi-miR160b were 23 bp, and of vvi-miR160c, d and e were 21 nt. There was one base difference between the overlapping of vvi-miR160 mature sequences. MiR160 precursor sequence alignment result showed that flanking sequence was variable but the mature sequence was similar. Moreover, the mature sequence of vvi-miR160c, d, and e were 21nt, it’s the same with ath-miR160 (Fig. 7A). Phylogenetic analysis of miR160 precursors revealed that vvi-miR160a and vvi-miR160b were clustered into one branch, while vvi-miR160c, d, and e were clustered into another branch, and vvi-miR160c closed to ath-miR160c (Fig. 7B). And the RNAfold software was used to predict the stem-loop secondary structures of vvi-miR160 family members according to their precursors, and the vvi-miR160c got the highest free energy(Fig. 7C). Small RNA sequencing detected vvi-miR160 at a moderate expression level in different sequencing samples, but there was no differential expression among samples (Fig. 7D). Quantitative analysis of the relative expression of vvi-miR160 precursors found that vvi-miR160c was the highest expression number, followed by vvi-miR160b, and the expression of vvi-miR160a precursor was not detected (Fig. 7E).