Dynamic Expression of miRNAs and Their Targets in the Response to Drought Stress of Grafted Cucumber Seedlings

Accepted Manuscript Title: Dynamic Expression of miRNAs and Their Targets in the Response to Drought Stress of Grafted Cucumber Seedlings Author: Li Chao-han, Li Yan-su, Bai Long-qiang, He Chao-xing, Yu Xianchang PII: DOI: Reference:

S2468-0141(16)00003-0 http://dx.doi.org/doi: 10.1016/j.hpj.2016.02.002 HPJ 2

To appear in:

Horticultural Plant Journal

Received date: Revised date: Accepted date:

25-7-2015 3-11-2015 15-11-2015

Please cite this article as: Li Chao-han, Li Yan-su, Bai Long-qiang, He Chao-xing, Yu Xianchang, Dynamic Expression of miRNAs and Their Targets in the Response to Drought Stress of Grafted Cucumber Seedlings, Horticultural Plant Journal (2016), http://dx.doi.org/doi: 10.1016/j.hpj.2016.02.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Dynamic Expression of miRNAs and Their Targets in the Response to Drought Stress of Grafted Cucumber Seedlings Li Chao-hana,b,*, Li Yan-sua,*, Bai Long-qianga, He Chao-xinga , Yu Xian-changa,** a

The Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China b

Horticultural Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China

Recieved date 25 July 2015; in revised form date 3 November 2015; accepted date 15 November 2015

Abstract Grafting of cucumber is widely used to improve growth and tolerance to biotic and abiotic stresses. MicroRNAs (miRNAs) regulate plant growth and development, and respond to various stresses through negative, post-transcriptional regulation of the expression of their target genes. We grafted cucumber (Cucumis sativus) scions onto pumpkin (Cucurbita moschata) rootstocks to study the molecular mechanisms of miRNA-mediated grafting-induced responses to drought stress. The relative expressions of 17 selected miRNAs and their predicted target mRNAs were detected by quantitative real-time PCR in leaves and roots of hetero-grafted cucumber seedlings (cucumber as scion and pumpkin as rootstock) and auto-grafted cucumber seedlings (cucumber as scion and rootstock) after 24 hours of 15% PEG6000 treatment. Compared with the expression in leaves of auto-grafted cucumber seedlings, the expressions of most miRNAs in the leaves of hetero-grafted seedlings changed dynamically: induced under normal conditions, and reduced after 3 h of drought stress, and then induced after 8 h and 24 h of drought stress. Similarly, compared with the expression in roots of auto-grafted cucumber seedlings, the expressions of most miRNAs in the roots of hetero-grafted cucumber seedlings changed dynamically: reduced under normal condition, and still reduced after 1 h, but induced after 3 h and 8 h, then reduced significantly after 24 h of drought stress. These results are useful for the functional analysis of miRNAs in the mediation of grafting-dependent drought tolerance. Keywords: cucumber; graft; drought; miRNA; target

1. Introduction As an important horticultural technique, grafting has been widely used to improve growth and resistance of plants to certain soil-borne diseases and abiotic stresses, including low soil temperatures (Schwarz et al., 2010), salinity (Estañ et al., 2005) and drought (García-Sánchez et al., 2007). Water-use efficiency can be improved by grafting scions onto rootstocks that have good tolerance to drought stress (García-Sánchez et al., 2007; Satisha et al., 2007). Previous studies demonstrated the mechanisms of graft-induced drought tolerance in bean (Phaseolus vulgaris) (Sanders and Markhart, 1992), soybean (Glycine max) (Serraj and Sinclair, 1996), and grape (Vitis vinifera) (Satisha et al., 2007). Grafted mini-watermelon onto a pumpkin rootstock obtained over 60% higher marketable yield under deficient irrigation conditions than that of ungrafted plants (Rouphael et al., 2008). Plants respond to or tolerate dehydration by altering photosynthesis, cell growth, or inducing the expressions of certain genes to maintain water uptake or cell turgor under drought stress (Chaves et al., 2008). Genes encoding functional proteins such as chaperones, LEA (late embryogenesis abundant) proteins and aquaporins, are induced under drought stress (reviewed by Yamaguchi-Shinozaki and Shinozaki (2006)). Additionally, many transcription factors, including MYB (Abe et al., 1997), AP2/ERF (Kizis et al., 2001), C2H2 zinc finger (Sakamoto et al., 2004), NAC (Lu et al., 2007), bZIP (Xiang et al., 2008) and WRKY families (Wang et al., 2009), are all involved in drought-responsive pathways. However, how the regulatory elements function together

*Contributed equally to this work as co-first authors. **Corresponding author: Tel: +86-10-82105980. Fax: +86-10-8210-9588. Email address: [email protected].

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requires further study. Plant miRNAs play critical roles in the regulation of plant growth (Mallory et al, 2004), flower development (Chen, 2004; Zhu et al., 2009), and responses to abiotic stresses, such as high salinity (Ding et al., 2009; Liu et al., 2008), high or low temperatures (Lv et al., 2010; Zhang et al., 2009), and deficiencies in phosphorus (Fujii et al., 2005; Pant et al., 2009) and nitrogen (Zhao et al., 2011). A group of miRNAs responsive to drought stress have been identified in rice (Oryza sativa) (Zhao et al., 2007; Zhou et al., 2010), Arabidopsis thaliana (Liu et al., 2008) and Medicago truncatula (Wang et al., 2011). Under drought stress, Nuclear Factor Y5 (NFY5), the target gene of miR169, was induced in vascular tissues and guard cells in Arabidopsis, while the expression of miR169 was suppressed (Liu et al., 2008). Overexpression of miR393 down regulated expression of its targets, the auxin receptors genes (TIR1 and AFB2), and led to more tillers, early flowering and more sensitivity to drought stress in rice (Xia et al., 2012). Overexpressing the rice miR319 gene in creeping bentgrass (Agrostis stolonifera) suppressed the expression of its target genes, increased leaf wax content and water retention, thus improving drought tolerance (Zhou et al., 2013). A detailed investigation of the miRNA-mediated response to drought stress would shed new light on the molecular mechanisms of graft-induced tolerance to drought. Recent studies found that grafting changed the expressions of several miRNAs. For example, the expressions of miR156/157 were dramatically suppressed by grafting in Citrus leaf petioles (Tzarfati et al., 2013). The expressions of more than 40 miRNAs were altered in the leaves of watermelon (Citrullus lanatus) grafted with bottle gourd (Lagenaria siceraria) or squash (Cucurbita maxima × Cucurbita moschata) (Liu et al., 2013). Previous studies showed that grafting could improve water use efficiency, and promote the growth and yield of cucumbers (Kong et al., 2012; Zhang et al., 2002). However, the mechanism of how miRNAs and their target genes mediate grafting-dependent drought tolerance is largely unknown. Here, eleven conserved miRNAs and six novel miRNAs, as well as their predicted target mRNAs, were selected according to the results of our previous study (Li et al., 2014) and their expression profiles were detected in grafted cucumber seedlings under drought stress. The results clarified the role of miRNAs’ response to drought stress in grafted cucumber seedlings.

2. Materials and methods 2.1 Plant materials and drought stress Healthy and uniform cucumber seeds (Cucumis sativus L., ‘9930’, supplied by the Institute of Vegetables and Flowers, Chinese Agricultural Academy of Science (IVF, CAAS), China) and pumpkin (Cucurbita moschata Duch. Ex Poir cv. ‘Jingxin No. 5 Rootstocks’, supplied by the National Engineering Research Center for Vegetables, China) were pre-germinated in an incubator, and then were planted in a solar greenhouse at the IVF, CAAS. The peg-grafting method was applied to construct hetero-grafted cucumber seedlings (cucumber scions grafted onto pumpkin rootstocks). The auto-grafted cucumber seedling was cut at the hypocotyl to avoid the effect of cutting on the seedlings. Uniform and healthy grafted seedlings were chosen. Every nine hetero- or nine auto-grafted seedlings were transplanted into a 60×15×15 cm trough for hydroponics with 0.5 strength Yamasaki cucumber nutrient solution (3.5 mM Ca(NO 3)2, 6 mM KNO3, 1 mM NH4H2PO4, 2 mM MgSO4, 0.1 mM Na2Fe-EDTA, 0.05 mM H3BO3, 10 μM MnSO4, 1 μM ZnSO4, 0.32 μM CuSO4 and 0.02 μM (NH4)6Mo7O24, pH 6.5). The nutrient solution was replaced every 5 days. The seedlings were transplanted into complete Yamasaki cucumber nutrient solution when the seedlings formed their third true leaves. When the seedlings formed their fourth leaves, the hetero- and auto-grafted seedlings were divided into two groups: (1) Drought-stressed group contains 36 hetero-grafted seedling and 27 auto-grafted cucumber seedlings, the seedlings were grown in complete Yamasaki cucumber nutrient solution containing 15% polyethylene glycol 6000 (PEG6000); (2) Non-stressed group contains 27 hetero-grafted seedling and 27 auto-grafted cucumber seedlings, the seedlings were grown in the complete nutrient solution. Non-stressed seedlings samples were set as 0 h. For drought-stressed cucumber seedlings, the third and fourth leaves counted from the basal part of stem were collected, and root tips were excised from rootstocks after 1, 3, 8 and 24 h of drought stress. The leaves and roots of non-stressed cucumber seedlings were collected at the same time points. The samples were cut into pieces quickly, placed into liquid nitrogen and stored at −80 C for RNA extraction. 2

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2.2 Verification of cucumber and pumpkin miRNAs and predicted target mRNAs by quantitative real-time PCR To detect the relative expression of the identified miRNAs, 11 known miRNAs and six novel miRNAs in cucumber and pumpkin were selected for quantitative real-time PCR (qRT-PCR) analysis. According to the regulatory role of the 17 miRNAs, they can be grouped as: (1) growth and development-related miRNAs; (2) stress resistance related miRNAs; and (3) miRNAs involved in miRNA homeostasis (Table 1). Using a miRcute miRNA isolation kit (DP501; Tiangen, Beijing, China), small RNAs were extracted and isolated from leaves and roots. Poly A tails were linked to the 3 of miRNAs, and first-strand cDNA was synthesized with a miRcute miRNA First-Strand cDNA Synthesis kit (KR201, Tiangen). A miRcute miRNA qPCR Detection kit (SYBR Green) (FP401, Tiangen) was used for qRT-PCR, and small U6 snRNA served as the internal reference. The qRT-PCR experiments were performed on an Mx3000PTM PCR system (Agilent-Stratagene, La Jolla, CA, USA). The program was set as: an initial 94 °C for 120 s; followed by 44 cycles of 94 C for 20 s and 60 C for 34 s; then a final dissociation/melt step of 95 C for 60 s, 55 C for 30 min, and 95 C for 30 s. Seventeen predicted target mRNAs of the 11 conserved miRNAs and six novel miRNAs were chosen for qRT-PCR detection. Primers were designed using cucumber mRNA sequences and Cucurbita pepo unigenes, and the EF1α gene (Elongation factor 1 gene) served as the internal reference. By applying an RNAprep Pure Plant Kit (DP432, Tiangen), total RNA was extracted from the same leaves or roots used for miRNA qRT-PCR detection. First-strand cDNA was synthesized with a FastQuant RT-Kit (KR106, Tiangen). A SuperReal PreMix Plus (SYBR Green) kit (FP205, Tiangen) was used for qRT-PCR. qRT-PCR experiments were performed on an Mx3000PTM PCR system, and the program conditions included an initial 95 C for 15 min; followed by 40 cycles of 95 C for 10 s and 60 C for 32 s; then a final dissociation/melt step of 95 C for 60 s, 55 C for 30 min, and 95 C for 30 s. All reactions were run in triplicate for each sample. Relative expression of the miRNAs and target genes was calculated by the comparative Ct (cycle threshold) method (2-ΔΔct). Differences between values were analyzed by Duncan’s multiple-range tests, with statistical significance set at P < 0.05.

3. Results 3.1 Expression of miRNAs in hetero- and auto-grafted cucumber seedlings under drought stress 3.1.1 Expression of miRNAs in leaves Compared with normal conditions, the expressions of most miRNAs in the leaves of cucumber seedlings were increased dynamically under drought stress (Fig.1, A). Compared with normal condition, in the leaves of hetero-grafted cucumber seedlings (CPL), the expressions of all miRNAs except b-miR-n07 were induced by 2 to 50 times after 1 h of drought stress. Expressions of most miRNAs were reduced after 3 h of drought stress than in normal condition. After 8 h of drought stress, except for the reduced expression of b-miR-n07, miR168 and b-miR-n02, the expressions of most miRNAs was induced. The expressions of most miRNAs in the leaves of auto-grafted cucumber seedlings (CCL) were induced remarkably during drought stress, especially the expression of most miRNAs were increased by 5 to 150 times after 1 h and 3 h of drought stress (Fig.1, B). 3.1.2 Expression of miRNAs in roots Compared with normal conditions, except for the induced expressions of b-miR-n10 and b-miR-n24, the expressions of most miRNAs in the roots of hetero-grafted cucumber seedlings (CPR) was reduced after 1 h of drought stress, and the expressions of most miRNAs was increased by 3 to 20 times after 3 h and 8 h of drought stress. After 24 h of drought stress, the expressions of most miRNAs were lower than that of 3 h and 8 h of drought stress, but still higher than under normal condition (Fig.1, C). Compared with normal condition, the expressions of most miRNAs in roots of auto-grafted cucumber seedlings (CCR) were increased by about 2-fold after 1 h of drought stress, while the expressions of miR159, miR168, and b-miR-n07 were repressed. Compared with normal condition, the expressions of most miRNAs were induced to some extents after 3 h and 8 h of drought stress, while induced dramatically after 24 h of drought stress (Fig.1, D).

3.2 Expression of target mRNAs in hetero- and auto-grafted cucumber seedlings under drought stress 3.2.1 Target mRNAs expression in leaves 3

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Compared with normal condition, expressions of most target mRNAs except TB7, TB10 and TB24 were induced to some extents in the leaves of hetero-grafted cucumber seedlings (CPL) during drought stress (Fig.2, A). Most target mRNAs showed induced expressions during drought stress in the leaves of auto-grafted cucumber seedlings (CCL). Moreover, after 3 h and 8 h of drought stress, expressions of all target mRNAs were remarkably higher than under normal condition (Fig.2, B). 3.2.2 Target mRNAs expression in roots Compared with normal conditions, in the roots of hetero-grafted cucumber seedlings (CPR), the expressions of seven target mRNAs (TB167, TB170, TB172, TB7, TB169, TB399, TB168, TB396, TB2) were reduced during drought stress, while TB159, TB319, TB10, TB24, TB395, TC19 and TB20 showed higher expressions at most time points of drought stress than under normal condition (Fig.2, C). Compared with normal condition, in the roots of auto-grafted cucumber seedlings (CCR), some target mRNAs such as T172, T319, TB7, T395, T398, T399, T396 and TB20 showed induced expression during drought stress than under normal condition, especially expressions of T172, T319, TB7, T398 and T396 were increased by over 5 folds after 3 h, 8 h and 24 h of drought stress (Fig.2, D).

3.3 miRNAs are involved in the response to drought stress in grafted cucumber seedlings To determine those miRNAs and targets that are responsive to drought stress in grafted cucumber seedlings, the expressions of miRNAs and targets in leaves (CPL) and roots (CPR) of hetero-grafted cucumber seedlings were compared with those in auto-grafted cucumber seedlings leaves (CCL) and roots (CCR). Under normal condition, the expressions of all miRNA except miR398 were induced by 2 to 5 times in the CPL/CCL. After 1 h of drought stress, the expressions of miR172, miR319, b-miR-n10, b-miR-n24, miR395 and miR168 was induced in the CPL/CCL. The expressions of the other miRNAs except b-miR-n07 were severely reduced in the CPL/CCL after 3 h of drought stress. While except b-miR-n07, b-miR-n02 and b-miR-n20, the expressions of most miRNAs in the CPL/CCL were increased after 8 h and 24 h of drought stress (Fig.3, A). Except the induced expressions of miR319, b-miR-n10, b-miR-n24, csa-miR-n19 and miR396, most miRNAs in the CPR/CCR were reduced under normal condition (0 h). In the CPR/CCR, the expressions of all miRNAs except b-miR-n07 and miR168 were remarkably reduced after 1 h of drought stress, while induced after 3 h of drought stress. The expressions of the most miRNAs except miR159, miR167, b-miR-n20, miR399 and b-miR-n02 in the CPR/CCR were induced after 8 h of drought stress. After 24 h of drought stress, the expressions of all miRNAs except b-miR-n24 in the CPR/CCR were lower than those after 1 h, 3 h or 8 h of drought stress, according to the CPR/CCR (Fig.3, B).

3.4 Target genes are involved in the response to drought stress in grafted cucumber seedlings. Like the comparison of miRNAs, according to the CPL/CCL, except for the dramatically induced expression of the targets of b-miR-n07, csa-miR-n19 and b-miR-n20, the expressions of most target genes did not change significantly under normal conditions (0 h). After 1 h of drought stress, the expressions of the targets of miR159, miR319, b-miR-n07, b-miR-n24, miR395, csa-miR-n19 and b-miR-n20 were induced in the CPL/CCL, while targets of miR167, miR170, miR172, b-miR-n10, miR169 and miR399 were reduced. After 3 h of drought stress, the expressions of most targets, except the targets of miR170, b-miR-n07, b-miR-n24 and csa-miR-n19, were increased in the CPL/CCL. After 8 h and 24 h of drought stress, the expressions of most target genes were reduced (Fig.3, C). Under normal condition (0 h), the expressions of most target genes, such as the targets of miR167, miR170, b-miR-n07, b-miR-n10, miR395, miR398, miR168, miR396 and b-miR-n20, were induced in the CPR/CCR. Under drought stress, the expressions of most targets, such as targets of miR170, miR172, b-miR-n07, b-miR-n10, miR395, miR398, csa-miR-n19, miR168, miR396 and b-miR-n20, were dramatically induced in the CPR/CCR, while targets of miR159, miR167, miR319, b-miR-n24, miR169, miR398, miR399 and b-miR-n02 were severely reduced (Fig.3, D).

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4. Discussion 4.1 Drought response of miRNAs and their target genes In the present study, the expression profiles of most miRNAs and their target mRNAs were altered under drought stress in leaves and roots of hetero- and auto-grafted cucumber seedlings (Fig.1, 2). According to the relationship of miRNAs and their targets, the stresses tolerance of plants would be improved by the increase in positive regulators or the decrease in negative regulators. Our results showed dynamic expression profiles of miR170, miR172 and miR395 at different time points of drought stress, which was different to the research of Zhou et al (2010). In our study, compared with normal conditions, the expression of miR169 was induced in both auto- and hetero-grafted cucumber seedlings under drought stress (Fig.1, A, B), while the expression of miR169 was reduced in Arabidopsis (Li et al., 2008) and Medicago truncatula (Wang et al., 2011) but induced in rice (Zhao et al., 2007; Zhou et al., 2010). In our study, the expression of miR398 was induced in the CCL and CPL under drought stress, but was suppressed in the CPR after 1 h of drought, while expression of miR398 was detected to be induced in Medicago truncatula (Trindade et al., 2010) and wild emmer wheat (Triticum turgidum) (Kantar et al., 2011) but reduced in maize (Wei et al., 2009). This discrepancy between our study and previous studies may be attributed to the differences of plant species, plant materials (general plant or grafted plant) and stress intensity of drought. In the present study, compared with normal condition, the negative expression changes between miRNAs and target mRNAs were exhibited in the CCR after 1 h of drought stress, and in the CPR after 1 h or 3 h of drought stress. Moreover, compared with auto-grafted cucumber seedlings, in the CPL, the expressions of most miRNAs were reduced after 3 h, and induced after 8 h and 24 h of drought stress, while the expression changes of most targets were the reverse (Fig.3, A, C). However, the negative correlation was not exhibited in the CPR under normal or drought stress (Fig.3, B, D). The reasons may be: (1) grafting changed the expression profiles of miRNAs and their targets; (2) only the miRNA of each family that has highest read count were chosen for qRT-PCR detection; (3) most miRNAs have more than one predicted target gene, but only one target of each miRNA was detected by qRT-PCR; and (4) there may be interspecific differences between cucumber and pumpkin; therefore the comparison of expression of miRNAs and targets between cucumber and pumpkin roots may not be valid. The dynamic expression of miRNAs in hetero-grafted cucumber seedlings indicated that they might be involved in regulation of drought response of grafted cucumbers. In addition, some miRNA are only responsive to drought stress, but not grafting or grafting combined with drought stress treatment.

4.2 Involvement of miRNAs -- targets regulatory network in grafting-dependent response to drought stress MiR168, miR396, b-miR-n02 and b-miR-n20 are predicted to target genes encoding Argonaute1 (AGO1), Dicer-like 1 (DCL1), Pre-mRNA-processing factor 17-like protein and Dicer-like 4 (DCL4), respectively. DCL1, DCL4, and AGO1 are involved in generation of miRNAs (Kurihara and Watanabe, 2004; Baumberger and Baulcombe, 2005; Rajagopalan et al., 2006). In our study, compared with auto-grafted cucumber seedlings, the expressions of miR168, miR396, b-miR-n02 and b-miR-n20 were reversed between the leaves and roots of hetero-grafted cucumber seedlings under normal conditions and drought stress (Fig.3, A, B). This might explain the dynamic changes in expression of other miRNAs and target genes. miR159, miR167, miR170, and miR172 together with their target genes (MYB, AFR8, GRAS, and APETALA2, respectively) are regulating plant growth and plant development (Llave et al., 2002; Achard et al., 2004; Chen, 2004; Zhu et al., 2009; Todesco et al., 2010; Liu et al., 2012). In the present study, the predicted target genes of b-miR-n07, b-miR-n10 and b-miR-n24 encode ATPase, GRAS transcription factor and DELLA protein, respectively. The function of b-miR-n07 may be similar to that of miR170 (Llave et al., 2002; Todesco et al., 2010). b-miR-n24 and DELLA might regulate the growth of roots and shoots through GA signaling pathway (Cheng et al., 2004). Thus, these miRNAs and targets may respond to drought by regulating vegetative and reproductive growth of grafted cucumber seedlings. Previous study demonstrated that miR169 and its targets, the NFY family, are involved in drought tolerance, and miR169 expression was induced in rice under drought stress (Zhou et al., 2010). Overexpression of NFY could enhance drought tolerance and improve yields in maize and Arabidopsis (Nelson et al., 2007). Similarly, the tolerance of NFY5- or NFY7-overexpressing Arabidopsis to drought stress also was increased by improving the water-use efficiency (Han et al., 2013). However, Arabidopsis 5

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overexpressing miR169a exhibited more severe water loss and decreased resistance to drought (Li et al., 2008). In our study, compared with auto-grafted cucumber seedlings, the expression of NFYA was reduced in the CPL and CPR at most time points during drought stress (Fig. 3, C, D), while the expression of miR169 was induced (Fig.3, A, B). Therefore, the induced expression of miR169 and the reduced expression of NFYA may be significant to enhance drought tolerance of cucumber. However, other studies showed that the expression of miR169 was reduced under drought stress (Li et al., 2008; Wang et al., 2011). Thus, miR169 may respond differently to drought in different plant species and under different intensities of drought stress. MiR395 responds to sulfate deficiency by targeting APS (Jones-Rhoades and Bartel, 2004). Overexpressing miR395 or suppressing APS might affect sulfate translocation and metabolism, and cause an imbalance of sulfate homeostasis (Kawashima et al., 2011). Previous studies demonstrated that plants can absorb more phosphorus after induction of miR399 or suppression of its target, ubiquitin E2 conjugase gene (UBC24), under Pi-deficiency conditions (Fujii et al., 2005). In our study, compared with the CCR, the expression of UBC24 was severely suppressed in the CPR, which indicated that Pi-uptake ability might also be enhanced in hetero-grafted cucumber seedlings. Unfortunately, the Pi and sulfate contents in the leaves and roots were not detected. MiR398 and its target, CSD1/2, participate in the response to oxidative and other stresses (Jagadeeswaran et al., 2009). Our results showed that the expression of CSD was remarkably induced in the CPR than in the CCR, indicating that, the roots of pumpkin rootstocks might enhance their drought tolerance through increased scavenging of ROS.

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Fig. 1 Expressions of miRNAs in hetero- and auto-grafted cucumber seedlings under drought stress Relative expression levels of miRNAs (compared with those under normal conditions) in auto- and hetero-grafted cucumber seedlings were calculated after 1, 3, 8 and 24 h of drought stress. Data are means ± standard error (SE). Expression levels of miRNAs in leaves of hetero-grafted seedlings (CPL; A), leaves of auto-grafted seedlings (CCL; B), roots of hetero-grafted seedlings (CPR; C), and roots of auto-grafted seedlings (CCR; D).

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Fig. 2

Expression levels of target mRNAs in hetero- and auto-grafted cucumber seedlings under drought stress

Relative expression levels of mRNAs (compared with those under normal conditions) in auto- and hetero-grafted cucumber seedlings were calculated after 1, 3, 8 and 24 h of drought stress. Expression levels of mRNAs in leaves of hetero-grafted seedlings (CPL; A), leaves of auto-grafted seedlings (CCL; B), roots of hetero-grafted seedlings (CPR; C), and roots of auto-grafted seedlings (CCR; D).

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Fig. 3 Expression of miRNAs and target mRNAs in hetero-grafted cucumber seedlings under normal condition and drought stress Relative expression of miRNAs and their target mRNAs (Compared with auto-grafted cucumber seedlings) of hetero-grafted cucumber seedlings were calculated after 0, 1, 3, 8 and 24 h of drought stress. Expression of miRNAs in leaves (CPL/CCL; A) and roots (CPR/CCR; B) of hetero-grafted cucumber seedlings and expression of target mRNAs in leaves (CPL/CCL; C) and roots (CPR/CCR; D) of hetero-grafted cucumber seedlings.

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Table 1 Seventeen known and novel miRNAs in cucumber and pumpkin, divided into three groups based on their regulatory roles Loci Regulatory roles

Growth and development

miRNA

Target Gene

for

CucumisLoci for Cucurbita

Target description sativus

pepo

miR159

T159

MYB protein 306-like

Csa002643

PU000183

miR167

T167

Auxin response factor 8-like

Csa019265

PU029234

miR170

T170

GRAS transcription factor

Csa017516

PU029211

miR172

T172

Floral homeotic protein APETALA 2-like

Csa010225

PU048074

miR319

T319

Transcription factor MYB75-like

Csa001869

PU128818

b-miR-n-07

TB7

ATPase

Csa003758

PU053245

b-miR-n10

TB10

GRAS transcription factor

Csa017132

PU000596

b-miR-n24

TB24

DELLA protein GAI1-like

Csa005232

PU023295

miR169

T169

Csa011911

PU050142

Nuclear transcription factor Y subunit Stresses response

A-1-like miR395

T395

ATP sulfurylase 1

Csa021552

PU029094

miR398

T398

Superoxide dismutase

Csa018002

PU039111

miR399

T399

Protein PHR1-LIKE 1-like

Csa011000

PU056789

csa-miR-n19

TC19

Pleiotropic drug resistance protein 2-like

Csa004670

PU128143

miR168

T168

Protein Argonaute 1A-like

Csa004392

PU107282

miR396

T396

Endoribonuclease Dicer homolog 1-like

Csa000411

PU045051

b-miR-n02

TB2

Pre-mRNA-processing factor 17-like

Csa000887

PU015813

b-miR-n20

TB20

Dicer-like protein 4-like

Csa016353

PU005625

Feedback regulation of miRNA homeostasis and gene expression

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