Messenger RNAs Mobile in Salix Matsudana Grafts Were in Association With Plant Rooting


 Messenger RNAs exchanged between scions and rootstocks of grafted plants seriously affect their traits performance. The study goals were to identify the long-distance mRNA transmission events in grafted willows using a transcriptome analysis and to reveal the possible effects on rooting traits. The results showed that the Salix matsudana variety 9901 has better rooting ability than YJ, which reasonably improved the rooting performance of the heterologous grafts 9901 (scion) / YJ (rootstock). A transcriptome analysis showed that 2,948 differentially expressed genes (DEGs) were present in the rootstock of 9901/YJ grafted plants in comparison with YJ/YJ. Among them, 692 were identified as mRNAs moved from 9901 scion based on a SNP analysis of two parents. They were mostly 1,001–1,500 bp, had 40–45% GC contents, or had expression abundance values less than 10. However, mRNAs over 4,001 bp, having 50–55% GC contents, or having expression abundance values of 10–20 were preferentially transferred. Eight mRNAs subjected to long-distance trafficking were involved in the plant hormone pathways and may significantly promote the root growth of grafted plants. Thus, heterologous grafts of Salix matsudana could efficiently influence plant rooting since of the mRNAs transport from scion to rootstock. Thus, the grafting parents and grafting patterns would be much concerned in the breeding process to gain the expected results in future.


Introduction
Grafting is used for the asexual propagation of woody plants, allowing the rapid proliferation and preservation of improved lines [1], as well as the shortening of owering plants' breeding periods, without altering genotypes [2].
Occasionally, grafting results in changes in plant traits. For example, Siberian pine (scion) grafted onto Scots pine (rootstock) (presented as scion/rootstock) grew faster and taller than Siberian/Siberian pines [1]. A wild-type (WT) tomato plant grafted on the mouse ear leaf mutant resulted in the scion leaves being phenotypically similar to the mouse ear mutant leaves [3]. The tomato grafts displayed leaf phenotype changes from the original multi-serrated angustifoliate leaves of the scion to the ovate leaves of the transgenic rootstock containing the Cmgaip gene (Cucurbita maxima) [4]. And, grafting a watermelon scion onto a Cucurbit rootstock effectively improved the plant's resistance to wilt disease [5], drought [6], cold [ 7], and heavy metal pollution [8].
Several studies had explored the possible mRNAs mobile mechanisms and revealed that the transcripts could travel long distances between scions and rootstocks, leading to variations in the grafted plants. For example, the CmGAI transcript has been readily detected in the WT scion grafted onto a CmGAI transgenic tomato rootstock [4]. In potato grafts, BEL5 transcripts from the BEL5-overexpressing scion have been found in the WT rootstocks [9]. The transcriptome was analyzed to determine the transcript compositions in heterologous grafts using single nucleotide polymorphisms (SNPs). Large numbers of mRNAs were identi ed as having undergone long-distance movement. In grape [10], 1,963 mRNAs were transferred from scions to rootstocks, while 2,210 mRNAs were transferred from rootstocks to scions.
Evaluations of the mobile mRNAs showed that highly expressed genes might generate great quantities of mobile mRNAs and that ~ 10% of the mobile mRNAs showed large differences in their transmission ratio owing to different eld conditions. This suggested the existence of complicated passive and selective mechanisms of mRNA movement. Genotypes, graft combinations and growth environments impacted the direction of the mRNA movement, as well as the numbers and species of mRNAs being exchanged. Transcriptome studies were also conducted in some other graft plants such as Arabidopsis thaliana (2,006 mobile mRNAs) [11], cucumber (3,546 mobile mRNAs) [12], and tobacco (1,163 mobile mRNAs) [13].
Salix matsudana is a widely planted willow species that has rich germplasm resources and plays an important role in greening, ecological protection, and conifer utilization. Grafting has been widely used in willow production. Here, the study goals were to identify the long-distance mRNA transmission events in grafted willows using a transcriptome analysis and to reveal the possible effects on rooting traits. The further characterization of transferred mRNA will provide insights into long-distance mobile mRNAs mechanisms and help improve grafting systems.
Results 9901 scion enhanced the rooting of YJ rootstock in 9901/YJ plants During hydroponic culturing, the grafted willows started rooting at the bases of the rootstocks. Obviously, the 9901/9901 grafts rooted faster than the YJ/YJ plants (Fig. 1). The total root length of the 9901/9901 grafts was 2.66 times that of the YJ/YJ by the 22nd d after grafting, indicating that 9901 has a strong rooting potential than YJ (Fig. 2). The 9901/YJ plants rooted faster than YJ/YJ plants, and the total root length of the 9901/YJ grafts by the 22nd d after grafting was 1.93 times that of the YJ/YJ grafts. This revealed that the 9901 scions, with a fast rooting potential, had a certain promotive effect on the rooting of the YJ rootstocks.

Degs In Homologous And Heterologous Willow Grafts
A total of 49,425, 48,176 and 48,713 genes were identi ed in the rootstock tissues of 9901/9901, YJ/YJ, and 9901/YJ grafted plants, respectively (Fig. 3), using a transcriptome analysis. In total, 2,948 DEGs were identi ed between YJ/YJ and 9901/YJ, which probably resulted in their different rooting performances. Among them, 1,687 and 1,261 genes were signi cantly up-and down-regulated, respectively, in 9901/YJ compared with in YJ/YJ.
A total of 2,309 DEGs (S1 Fig) were annotated in the GO database. The DEGs related to biological processes were grouped in 21 terms, which were mainly involved in metabolic (1,100 genes), cellular (983 genes), and single-organism (916 genes) processes. The DEGS related to cellular component were grouped in 15 terms, which were mainly involved in cell (999 genes), cell part (975 genes), membrane (931 genes), and organelle (571 genes). The DEGS related to molecular function were grouped in 15 terms, which were mainly involved in catalytic activity (1,226 genes), binding (1,170 genes), and transporter activity (216 genes). SNP analysis revealed 692 shoot-to-root mobile mRNAs in the 9901/YJ graft SNP analysis was conducted between the homologous genes of two grafts (YJ/YJ, 9901/YJ), which showed 692 of 48,713 mRNAs in rootstock of 9901/YJ came from the scion. Among them, 536 mRNAs were annotated in the GO database (Fig. 4). In total, 16 annotated terms were related to biological processes, including metabolic processes (253 mRNAs), cellular processes (234 mRNAs), and singleorganism processes (163 mRNAs). There were 14 annotated terms related to cellular component, including cell (240 mRNAs), cell part (239 mRNAs), and membrane (201mRNAs), and there were 11 annotated terms related to molecular function, including binding (301 mRNAs) and catalytic activity (259 mRNAs).
An example was given in detail. The ABF (ABA responsive element binding factor) (K14432) gene was 966 bp in both the parents but displayed three SNPs at site of 135, 200, and 259 bp in the grafted plants (Fig. 5). The SNPs resulted in coding sequence changes in two amino acids, which further affected the secondary structure of the 9901-sourced homologous protein having less alpha helix and extension chain in comparison with the YJ-sourced protein, even far from the SNP site (Fig. 6). The polymorphisms in the proteins might really contribute to graft performance.

Characterization Of The Mobile Mrnas In Grafted Willows
The transfer ratio of 455 mobile mRNAs were estimated (the others, having less than 30 reads, were not considered), and they ranged from 7.14-68.75% with an average of 27.30%. Among them, 29.89%, 23.74%, and 20.88% of the mRNAs had transfer ratio of 10-20%, 20-30%, and 30-40%, respectively.
The maximum size of a mobile mRNAs was 10,710 bp and the minimum was 186 bp, with an average of 1,657 bp, which was 19.64% greater than the average 1,385 bp length of all willow transcripts. On the basis of mRNA length, the 455 mobile mRNAs were classi ed into several groups. The 1,000-1,500bp group contained the largest proportion, at 26.59%, of all the mobile mRNAs, followed by the 501-1,000 bp and 1,501-2,000 bp groups. However, a transfer ratio analysis revealed that mRNAs > 4,001 bp were preferred for transfer, accounting for 2.40% of the mobile mRNAs in the group, followed by the 3,501-4,000 and 2,501-3,000-bp mRNAs (Table 1).  Table 2). Among them, the mRNAs with GC contents of less than 35% were not transferred. The highest proportion, at 64.18%, of all the mobile mRNAs had GC contents of 40-45%, followed by mRNAs with GC contents of 45-50% and 50-55%. The transfer ratio calculation showed that the mRNAs with GC contents of 50-55% had the largest value at 1.20%, followed by the mRNAs with GC contents of 40-45% and 35-40%. The expression abundances (presented as FPKM values) of the mobile mRNAs (Table 3)   In comparison with the previous reports, several mobile mRNAs were recognized in association with rooting such as ABCB29 [14], ARR5 [15], ERF9 [16], and ABF [17]. PCR tests showed them well expressed in 9901 rootstock of 9901/9901 plants, but less in YJ rootstock of YJ/YJ plants (Fig. 7). Obviously, these genes had more transcripts in YJ rootstock of 9901/YJ plants than in YJ rootstock of YJ/YJ plants, potentially gained from its 9901scion and resulted in their improved rooting ability.
KEGG pathways of the mobile mRNAs participate in root formation A total of 142 (20.52%) mobile mRNAs were annotated to 85 pathways in the KEGG database. The main pathways included spliceosome, biosynthesis of amino acids, plant-pathogen interaction, and protein processing in endoplasmic reticulum (Table 4), which enriched with 11, 11, 10, and 10 mobile mRNAs, respectively.   (Table 5). They may directly or indirectly participate in root formation as previously reported.  [19], and 23 mobile mRNAs were identi ed in Canola /Arabidopsis grafts [13]. Thus, the numbers of the mobile mRNAs is correlated with the a nity between the scion and rootstock. Grafts involving two closely related varieties of a species could transfer more mRNAs than grafts involving more distantly related species. Our study here contributed the further evidence that more that 692 mobile mRNAs were identi ed in willow grafts, while both willow parents were bred from the same species of Salix matsudana.
Furthermore, the numbers of mobile mRNAs might also associate with the tissue conditions, tissue ages [10], and the sampling distance from the grafting site as the previous reports [11,12,13].

The Mobile Mrnas E ciently Affacted Willow Grafts' Rooting
Long-distance mRNA movement events in grafts really affect plant traits performance. Here, the willow graft experiment showed that 9901 scion transmitted its strong rooting ability to the YJ rootstock, thereby improving rooting by as much as 1.93 times that of YJ/YJ. Some knid of hormones were frequently involved in this process and convinced in this study. For example, AUX/IAA may activate the downstream transcription factor ARF, which is involved in regulating root development [20,21,22]. Interestingly, the AUX/IAA gene was found mobile in various grafts [9,23,24]. In willow, its transfer ratio reached 74.41%. A cellular mechanism for the positive role of ABA in promoting root meristem maintenance and root growth has been demonstrated in Arabidopsis. Two ABF genes in the ABA metabolic pathway were determined mobile in willow grafts at ratio of 1.91% and 15.24%, respectively. An amino acid analysis showed their structures varied in the two parents, which might have resulted in their different rooting e ciencies. As a transcription factor, the ABF gene regulates downstream gene expression, such as basic leucine zipper transcription factor of bZIP4 in maize (Zea mays). The ZmbZIP4 over-expression lines have longer primary, seminal, and lateral roots in comparison with those of the WT plants. Immunoprecipitation sequencing revealed that ZmbZIP4 positively regulates some target genes related to root growth, including ZmLRP1, ZmSCR, ZmIAA8, ZmIAA14, ZmARF2, and ZmARF3 [25]. Additionally, the ABF2 activity levels in the radicles and shoot meristem regions of ABF2 transgenic plants' embryos are high. During subsequent seedling growth, the highest ABF2 activity level, except the levels in the meristem regions and the elongation zones, was detected in lateral roots [26]. Thus, the mobile of the ABF gene in willow grafts may affect the rooting abilities of the grafts.
The mobile mRNAs in grafts are of some sequence characteristics An association analysis showed that the mRNAs characteristics, such as mRNA length, GC content, and mRNA expression abundance, had effects on the mobile e ciency in grafts. Several studies, such as those in Arabidopsis, N. benthamiana, cucumber, grape, and willow, revealed that more than 70% of the mobile mRNAs in grafts were less than 2,000 bp. However, transfer ratio analyses revealed that larger mRNA fragments were preferentially transferred in heterologous grafts. For example in willow, the transfer ratio of mRNAs > 4,000 bp was 2.4%, which is 3.13 times that of mRNAs < 2,000 bp. In Arabidopsis, the transfer ratio of mRNAs > 4,000 bp reached 11.52%, which was 2.55 times that of mRNAs < 2,000 bp. Probably, the larger mRNA fragments may easily form certain stable spatial structures and suited for long-distance transfers.
A previous analysis using a computational diffusion-based model showed that mRNA abundance is an important factor in a mRNA's long-distance mobile [27]. Unfortunately, the correlation between mRNA expression abundance and the transfer ratio was not perfectly de ned in several experiments, For example, in the willow grafts, mRNAs with the expression abundances of 10-20 and 40-50 had high transfer e ciencies, being 1.62 and 1.61 times that of mRNAs with expression abundances of 30-40. In N. benthamiana grafts, only 3 of 183 mobile mRNA were highly ranked among all the transcribed genes on the basis of their expression abundance levels [13]. In the over-expression experiments, the overexpressed GUS and GFP genes under control of the 35S promoter [28,29], and even AtAMT1 and AtCHL1 under the control of the strong phloem-speci c promoter SUC [13], did not undergo long-distance transfer. Thus, the effect of gene expression level on their mobile ratio in grafts might be in combination with some other mechanisms.
GC content is another factor that may in uence mRNA mobile. In willow grafts, 95.16% mobile mRNAs had GC content between 30% and 50%. Among them, mRNAs with GC content of 50-55% were preferentially transferred with a transfer ratio of 1.20%, which was 1.60 times that of mRNAs with GC contents greater than 55% and 1.13 times that of mRNAs with GC contents less than 50%. Similarly, mRNAs with GC contents of 50-55% exhibited the highest transfer ratio of 8.45% in Arabidopsis grafts, which is 1.08 times that of mRNAs with GC contents > 55% and 1.54 times that of the mRNAs with GC contents < 50%. Therefore, the mRNA mobile in grafts is of complicated mechanism, in which sequence characteristics was much concerned and probably was of comprehensive effect. More details will be explored in the future.

Plant materials and grafting
The current shoot segments of the Salix mandshurica variety 9901 and Yanjiang (YJ) were used as sources to construct the homologous and heterologous grafts 9901/9901, YJ/YJ, and 9901/YJ by Vshaped grafting method. The grafted segments were grown in hydroponics condition (changed every other day) in a growth chamber of a 16/8 h light/dark cycle (2000lux) and relative humidity of 75-80% at 25°C. The total root length was measured for each plant starting from 10th day of grafting. Permission for collection, planting and grafting of the willow plants was obtained from Beijing Forestry University.

Extraction Of Total Rna
At 22th day of grafting, the bark tissue (containing phloems for organic substances transporting) of 9901/9901, YJ/YJ and 9901/YJ rootstocks were individually sampled for total RNA extraction by using polysaccharide polyphenol kit (TIANGEN company). The isolated RNA was treated with RNase-free DNase I (Takara) for 30 min at 37°C to remove the residual DNA. RNA quality was tested by using NanoDrop2000 spectrophotometer (NanoDrop, Thermo Scienti c, Waltham, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).

Rna Library Construction And Sequencing
RNA sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer's instruction. The library was sequenced on an Illumina platform (Biomaker company) and the paired-end reads were generated. The clean data were obtained by removing adapter and low quality reads from the raw data. Q20, Q30, GC-content and sequence duplication level of the clean data were calculated. All the analyses were based on the high quality clean data.
Identi cation of the differentially expressed genes (DEGs) and function analysis The reads per kilobase per million mapped reads (RPKM) were generated to represent the expression abundance of each mRNA. Differential expression analysis of two data sets (YJ/YJ and 9901/YJ) was performed using the edgeR. The FDR < 0.01 & Fold Change ≥ 2 were as the threshold for signi cantly differential expression. Gene Ontology (GO) enrichment analysis of the DEGs was implemented by the GOseq R packages based Wallenius non-central hyper-geometric distribution, which can adjust for gene length bias in DEGs [30]. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using Blast2GO software (BioBam Bioinformatics SL) [31].

Determination Of The Transfer Rnas
All the clean reads of 9901/9901, YJ/YJ and 9901/YJ were compared by using HISAT2 software [32].
SNPs between two parents were identi ed with GATK software [33]. The reads (transcripts) in rootstocks of 9901/YJ having SNPs with YJ/YJ but homozygous with 9901/9901 were determined as the mobile mRNAs from 9901 scion to YJ rootstock in 9901/YJ grafts.

Estimation Of Transfer Ratio
Based on the expression abundance of the mobile mRNAs in 9901/YJ, the mobile mRNA transfer ratio from 9901 to YJ in 9901/YJ was estimated: [the normalized number of the 9901-sourced mobile mRNAs in the rootstock of 9901/YJ divided by the total normalized number of the 9901-sourced and YJ -sourced mobile mRNAs in the rootstock of 9901/YJ]. When SNPs were present in several mobile reads, the average value was used as the transfer ratio of the mobile mRNA. The mobile mRNA with less than 30 mapped reads in the graft was not considered for transfer ratio estimation so as to reduce the bias.

Declarations
Author contributions Yin Peng and Xu Jichen wrote the main manuscript text. Yin Peng, Liu Xiao and Lan Baoliang prepared Figs. 1-7. Yin peng, Cui Yu and Wang Yan prepared tables 1-5. All authors reviewed the manuscript. The total root length of willow grafts in hydroponics culture Page 16/19

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