Here we present a detailed protocol for efficiently making homo- and heterografts between watermelon and bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis, for the investigation of cold-responsive microRNAs.
MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20 – 24 nt, known to play important roles in plant development and adaptation. There is an accumulating evidence showing that the expressions of certain miRNAs are altered when grafting, an agricultural practice commonly used by farmers to improve crop tolerance to biotic and abiotic stresses. Bottle gourd is an inherently climate-resilient crop compared to many other major cucurbits, including watermelon, rendering it one of the most widely used rootstocks for the latter. The recent advancement of high-throughput sequencing technologies has provided great opportunities to investigate cold-responsive miRNAs and their contributions to heterograft advantages; yet, adequate experimental procedures are a prerequisite for this purpose. Here, we present a detailed protocol for efficiently generating homo- and heterografts between the cold-susceptible watermelon and the cold-tolerant bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis. The presented methods are also useful for other plant-grafting systems, to interrogate miRNA regulations under various environmental stresses, such as heat, drought, and salinity.
Grafting has long been employed as an agricultural technique to improve crop production and tolerance to biotic and abiotic stresses1,2,3. In heterografting systems, elite rootstocks can enhance water and nutrients uptake of plants, strengthen resistance to soil pathogens, and limit the negative effects of metal toxicity4,5, which may confer the grafts an enhanced growth vigor and increased tolerance to environmental stresses. In many cases, heterografting can also impact fruit qualities in horticultural plants, leading to improved fruit flavor and increased content of health-related compounds6,7. It has been found that the long-distance transfer of phytohormones, RNAs, peptides, and proteins between the rootstock and the scion is a fundamental mechanism modulating the growth and development reprogramming of scion plants8,9,10. Grafting has been widely used in studies of long-distance signaling and transport in relation to environmental adaptation11. Grafting experiments are especially powerful for unambiguous detection of transmitted molecules in receiving tissue or vascular sap, and activation or suppression of molecular targets due to signal transmission12.
Non-coding RNAs, a big class of RNA that exert important regulatory functions in cells, have been reported to play a role in facilitating plant adaptation to abiotic stress13. miRNAs are endogenous small non-coding RNAs of about 20 – 24 nt. Studies have revealed the regulatory role of miRNAs in various aspects of plant activities, such as shoot growth, lateral root formation14,15,16, nutrient uptake, sulfate metabolism and homeostasis17, and responses to biotic and abiotic stress18. Recently, the expression of miRNAs and their target genes were related to salt stress tolerance in heterografted cucumber seedlings19. In the intervariety grafts of grape, the responses of miRNA expression to drought stress were found to be genotype-dependent20.
The rapid development and decreasing cost of high-throughput sequencing technology have provided a great opportunity for the study of miRNA regulations in agronomical plants. Watermelon (Citrullus lanatus [Thunb.] Mansf.), an important cucurbit crop grown throughout the world, is susceptible to low temperatures. Bottle gourd (Lagenaria siceraria [Molina] Standl.) is a more climate-resilient cucurbit commonly used by farmers to graft with watermelon. The primary goal of the current study is to establish a standard, efficient, and convenient method for making heterografts between watermelon (Citrullus lanatus [Thunb.] Mansf.) and bottle gourd (Lagenaria siceraria [Molina] Standl). This protocol also provides a detailed experimental scheme and analytical procedures for the study of the regulation of miRNA expressions following grafting, which is useful for revealing the mechanisms underlying heterografting advantages.
The plant materials used in this study include the watermelon cultivar and the bottle gourd landrace. Watermelon cultivar is a commercial cultivar with high yield but susceptible to low temperatures. Bottle gourd landrace is a popular rootstock for grafting with watermelon, cucumber, and bottle gourd, due to its excellent tolerance of low temperatures21.
1. Seed Sterilization and Germination
2. Seedling Growth and Grafting
Figure 1: Illustration of the graft combinations and the grafted plant structures. WB = watermelon/bottle gourd heterografting; WW = watermelon/watermelon homografting; BB = bottle gourd/bottle gourd homo-grafting; WB-S = scion leaves of the watermelon/bottle gourd heterografts that were sampled; WB-R = rootstock leaves of the watermelon/bottle gourd heterografts that were sampled. Please click here to view a larger version of this figure.
3. Postgrafting Management, Cold Treatment, and Sampling
4. Library Preparation and High-throughput Sequencing
5. miRNA and Target Gene Prediction
6. Differential Expression and Gene Ontology Analysis
Figure 2: Phenotypes of various grafts at room temperature and cold-stressed conditions. (a) This panel shows homo- and heterografted seedlings at room temperature as the control. (b) This panel shows homo- and heterografted seedlings after 48 h of cold treatment. Please click here to view a larger version of this figure.
Using the described method, we obtained a high success (survival) rate of 98% for grafting. Phenotypes of various grafts at room temperature and cold-stressed conditions are shown in Figure 2. After 48 h of cold treatment, the homografted watermelon plants showed obvious growth retardation with wilted young leaves, while the homografted bottle gourd plants and the watermelon/bottle gourd heterografts exhibited much more vigorous growth. No symptoms of damage were observed in the leaves of the heterografts, which even outperformed the homografted bottle gourd plants, where the lowest true leaves were damaged. These results clearly demonstrate the advantage of heterografts in conferring cold tolerance.
Small RNA sequencing of the eight libraries yielded a total of 258 million raw reads. After the quality control (QC), a total of 146 million reads corresponding to approximately 30 million unique sequences were retained (Table 1). Based on this set of clean sRNA sequences, 323 miRNAs, including 10 known and 313 novel miRNAs, were predicted from the bottle gourd, and 20 known and 802 novel miRNAs were predicted from the watermelon.sRNAs of 24 nt made up the biggest class of sRNAs in all grafting combinations, regardless of room temperature or cold-stressed conditions (Figure 3).
Treatment | Code | No. reads | sRNA | |
Total | Unique | |||
WW-CK | Raw | 30612962 | ||
Clean | 19727501 | 3858868 | ||
Mapped to genomic | 19059359 | 3777952 | ||
BB-CK | Raw | 30845546 | ||
CK | Clean | 16832061 | 3787866 | |
Mapped to genomic | 16375142 | 3694388 | ||
WB-CK-S | Raw | 39492123 | ||
Clean | 26783053 | 6319473 | ||
Mapped to genomic | 25919944 | 6132389 | ||
WB-CK-R | Raw | 23763619 | ||
Clean | 10187791 | 1784447 | ||
Mapped to genomic | 8946929 | 1537867 | ||
WW-CL | Raw | 27557577 | ||
Clean | 17879038 | 3336242 | ||
Mapped to genomic | 17153763 | 3259960 | ||
BB-CL | Raw | 29780991 | ||
Clean | 13342206 | 3235570 | ||
Cold | Mapped to genomic | 12949972 | 3164329 | |
WB-CL-S | Raw | 45708415 | ||
Clean | 23071845 | 4310276 | ||
Mapped to genomic | 22363113 | 4224166 | ||
WB-CL-R | Raw | 30585408 | ||
Clean | 19029266 | 3541729 | ||
Mapped to genomic | 17364239 | 3196106 |
Table 1: Statistics of small RNAs in various grafts at room temperature or under cold treatment.
Figure 3: Size distribution of the sRNA reads in various grafts. (a) This panel shows the size distribution of the sRNA reads in heterografts under control or cold conditions. (b) This panel shows the size distribution of the sRNA reads in homografts under control or cold conditions. Please click here to view a larger version of this figure.
Upon a 48-h of cold treatment, 30 and 268 miRNAs were up- and downregulated, respectively, in the leaves of the scion in the heterografts. This was in sharp contrast to the results in the leaves of rootstock, where 31 and only 12 miRNAs were up- and downregulated, respectively (Figure 4). In the watermelon/watermelon homografts, 64 and 83 miRNAs were up- and downregulated, respectively. In the bottle gourd/bottle gourd homografts, these numbers were 30 and 28. Apparently, heterografting caused a profound reprogramming of the miRNA expressions. GO-enrichment analyses of the putative target genes of the differentially expressed miRNAs identified 78 enriched GO terms in the scion of the heterografts, with 40 classified into biological processes, 2 into cellular components, and 36 into molecular functions (Figure 5). We found that several known GO terms/pathways related to abiotic/biotic stress resistance and signal transduction, for instance, the chitin catabolic process (GO: 0006030, GO: 0006032), ethylene-activated signaling pathway (GO: 0009873), polyamine biosynthetic process (GO:0006596), and signal transduction by protein phosphorylation (GO: 0009755), were involved. Combined, our results suggest that the downregulation of miRNAs,by tuning the abundance of the transcripts of their target genes, may represent an important mechanism underlying enhanced cold tolerance. In the watermelon/bottle gourd grafts, the heterograft per se has a significant impact on the miRNA patterns that form the graft advantages.
Figure 4: Comparison of the patterns of up- and downregulated miRNAs in response to cold stress in various grafts. WB-S = scion leaves of the watermelon/bottle gourd heterografts that were sampled; WB-R = rootstock leaves of the watermelon/bottle gourd heterografts that were sampled; WW = the watermelon/watermelon homografts; BB = the bottle gourd/bottle gourd homografts. Please click here to view a larger version of this figure.
Figure 5: GO enrichment analyses of the putative target genes of differentially expressed miRNAs in the scion leaves of heterografts upon cold stress. WB-CL-S = scion leaves of the watermelon/bottle gourd heterografts under cold treatment; WB-CK-S = scion leaves of the watermelon/bottle gourd heterografts at room temperature. Please click here to view a larger version of this figure.
In this protocol, we described in detail a highly efficient and reproducible method to make homo- and heterografts between watermelon and bottle gourd. This method, requiring no specific equipment, is very easy to operate and typically has a very high survival rate of grafting. The method can also be used to make grafts for other cucurbits, such as between watermelon, cucumber, and pumpkin.
It is worth noting that the relative size (age) of the rootstock and scion is critical to making a successful graft (step 2.2 of the Protocol). We observed that, if the rootstock used was too large compared to the scion, the graft union was more difficult to form because the stem of the scion was somewhat hollowed. Based on our previous proteomic data31, the inclusion of self-grafted scion and self-grafted rootstock as controls is strongly recommended (step 2.3 of the Protocol), because then, the impact of grafting injuries can be largely eliminated.
This protocol also provides a detailed experimental scheme and specific experimental procedures for investigating the abundances of miRNAs in the heterografting system. This method will also be useful for studies in other plant-grafting systems to reveal the mechanisms of local and long-distance miRNA regulation. In the Representative Results, we report the expression changes of only local miRNAs in the scion or rootstock in response to a low temperature. Accumulating reports have highlighted the involvement of long-distance small RNA transmission in grafting-related phenotypic changes. The protocol presented here, which combines the methods for grafting and high-throughput data analysis, can also be used for miRNA transmission analysis between the scion and the rootstock. The principle for differentiating transmitted miRNAs from local miRNAs is based on their sequence similarity to the reference genomes (i.e., a miRNA in the scion that is more like the rootstock genome is considered to be transferred from the rootstock, and vice versa).
The authors have nothing to disclose.
This work was supported by the National Natural Science Foundation of China (31772291), the Research Project for Public Interest in Zhejiang Province (2017C32027), the Key Science Project of Plant Breeding in Zhejiang (2016C02051), and the National Program for the Support of Top-notch Young Professionals (to P.X.).
TRIzol Reagent | Invitrogen | 15596026 | |
RNA-free DNase I | Takara | D2270A | |
Truseq Small RNA sample prep Kit | Illumina | RS-200-0012 | |
2100 Bionalyser | Agilent | 5067 | |
DNA Polymerase | Thermo Fisher Scientific | F530S | |
UEA sRNA workbench 2.4-plant version (software) | NA | NA | http://srna-workbench.cmp.uea.ac.uk/ |
Rfam 11.0 database (website) | NA | NA | http://rfam.janelia.org |
miRBase 22.0 (website) | NA | NA | http://www.mirbase.org/ |
MIREAP(software) | NA | NA | https://sourceforge.net/projects/mireap/ |
TargetFinder (software) | NA | NA | http://targetfinder.org/ |