Skip to main content
Download PDF

Genome-wide screening of the RNase T2 gene family and functional analyses in jujube (Ziziphus jujuba Mill.)

BMC Genomics volume 24, Article number: 80 (2023) Cite this article

Abstract

Background

Ribonuclease (RNase T2) plays crucial roles in plant evolution and breeding. However, there have been few studies on the RNase T2 gene family in Ziziphus jujuba Mill., one of important dried fruit tree species. Recently, the released sequences of the reference genome of jujube provide a good chance to perform genome-wide identification and characterization of ZjRNase gene family in the jujube.

Results

In this study, we identified four members of RNase T2 in jujube distributed on three chromosomes and unassembled chromosomes. They all contained two conserved sites (CASI and CASII). Analysis of the phylogenetic relationships revealed that the RNase T2 genes in jujube could be divided into two groups: ZjRNase1 and ZjRNase2 belonged to class I, while ZjRNase3 and ZjRNase4 belonged to class II. Only ZjRNase1 and ZjRNase2 expression were shown by the jujube fruit transcriptome analysis. So ZjRNase1 and ZjRNase2 were selected functional verification by overexpression transformation of Arabidopsis. The overexpression of these two genes led to an approximately 50% reduction in seed number, which deserve further attention. Moreover, the leaves of the ZjRNase1 overexpression transgenic lines were curled and twisted. Overexpression of ZjRNase2 resulted in shortened and crisp siliques and the production of trichomes, and no seeds were produced.

Conclusion

In summary, these findings will provide new insights into the molecular mechanisms of low number of hybrid seeds in jujube and a reference for the future molecular breeding of jujube.

Peer Review reports

Background

RNA depolymerase, also known as ribonuclease (RNase), is a type of acidic endonuclease that belongs to the RNase T2 family [1]. In 1959, RNase T2 was identified in Aspergillus fungi [2]. Recent studies showed that S-glycoproteins in tobacco are responsible for self-incompatibility and are highly homologous to RNase T2 and RNase Rh from Rhizopus niveus [3,4,5,6,7]. S-glycoproteins have RNase activity and RNase T2 family members have two conserved active-site fragments (CASI and II) [7]. These findings indicated that the function of RNase T2 family members is closely related to plant gametophytic self-incompatibility, and the RNase T2 genes have been highly conserved throughout evolution. These discoveries could promote researches on the mechanism of plant self-incompatibility.

An increasing number of RNase T2/S-RNase enzymes have subsequently been discovered in the genomes of plants in Acacia, Solanaceae and Rosaceae [3, 8,9,10]. Among them, RNases were found in Japanese pear (Pyrus) and were shown to be involved in self-incompatibility of gametophytes [11]. The S-RNase genes of apricot (Prunus armeniaca) and loquat (Eriobotrya japonica) have also been identified as being related to gametophytic self-incompatibility [12, 13]. Thus, the RNase T2/S-RNase enzymes are closely related to gametophytic self-incompatibility and should be widely studied to increase the efficiency of hybrid breeding. Furthermore, it has been found that RNases is involved in abiotic stress responses such as salt stress, phosphate starvation and senescence [14].

Jujube (Ziziphus jujuba Mill.) a deciduous fruit tree species native to China, has been distributed worldwide [15,16,17,18]. Low fruiting set and seed production were key obstacles for hybrid creation in jujube cross breeding and no hybrid cultivar was successfully utilized in cultivation [18]. The exploration of RNase T2 family members and functions will be helpful for jujube cross breeding. However, the work on jujube RNase family has yet not been conducted, and the functions of ZjRNase are still unclear. The aim of this study is to identify the RNase T2 gene family in jujube genome and demonstrate the potential function of RNase T2 genes. The results provided new insights into the molecular mechanisms of low number of hybrid seeds in jujube and found new functions of RNase T2 family members in leaf and fruit development.

Results

Genome-wide identification of RNase T2 family members

Eight RNases have been identified in Oryza sativa [1]. The identification of jujube RNase T2 was performed via BLASTP searches and the Oryza sativa RNases protein sequences were used as query sequences to search the Ziziphus jujuba genome [19]. A total of 4 ZjRNases were identified by two rounds of BLASTP and conserved domain predictions (Table 1). The protein length from ZjRNase1 to ZjRNase4 is 226,280,242 and 158 and their encoding genes are located on chromosomes Chr1, Chr9, Chr10 and ChrUn, respectively. The predicted pIs ranges from 5.12 to 7.81, and the molecular weight is between 18.61 and 31.08 kDa. The instability index analysis showed that ZjRNase1, ZjRNase3 and ZjRNase4 are stable, except for ZjRNase2.The higher the aliphatic index was, the more stable the protein: among the jujube RNase T2 family members. ZjRNase3 is the most stable one, followed by ZjRNase4, ZjRNase2 and ZjRNase1. The ZjRNase grand average of hydrophobicity is negative, indicating that the proteins were slightly hydrophobic.

Table 1 Putative RNases in Ziziphus jujuba
Full size table

Phylogenetic analysis and multiple sequence alignment of ZjRNases

To better determine their evolutionary relationship and facilitate the classification of ZjRNases, a phylogenetic tree was constructed comprising the sequences of 14 MdRNases, 9 PyRNases and 4 ZjRNases (Fig. 1). The 4 ZjRNases were divided into two types: class I and class II (Fig. 1). ZjRNase1and ZjRNase2belongs to class I subgroups I a and I b, ZjRNase3 and ZjRNase4 belong to class II.

Fig. 1
figure 1

Rooted phylogenetic tree representing relationships between RNase proteins of Pyrus, Malus domestica and Ziziphus jujuba. All the RNase proteins were divided into three classes, class I was divided into two subgroups, which were represented by different coloured clusters. The phylogenetic tree was constructed by the NJ method using MEGA 7 software with 1000 bootstrap replicates

Full size image

Previously reported RNases have two conserved active sites, CASI and CASII motifs [20]. All the ZjRNases have the same conserved structural sites as the RNases in other plant species, suggesting that the four ZjRNase members we obtained in jujube are correct (Fig. 2). According to secondary structure analysis, it was predicted that α-helix or β-sheet structures are present at 14 positions, of which 7 (50%) may be α-helix structures (red) and 5 (35.71%) may be β-sheet structures (light green). The remaining two predictions were inconsistent and were classified as uncertain types. These results showed that the secondary structure of the ZjRNase member proteins are relatively stable.

Fig. 2
figure 2

Multiple sequence alignment of the conserved active sitesof ZjRNases. The alignment was constructed by MUSCLE and visualized by Jalview. The two conserved active site (CAS red thick box) regions were indicated. The protein secondary structures were predicted using HMM and PSSM software. The red boxes represent α-helices, and the light green boxes represent β-sheet

Full size image

Analysis of the structural and conserved motifs of ZjRNases

The results of further analysis of the gene structure and motifs of the ZjRNases were shown in Fig. 3. The phylogenetic tree confirmed that ZjRNases could be grouped into two classes (Fig. 3a). Analysis of the genomic DNA sequences showed that ZjRNases usually have 1, 2 or more than 2 introns (Fig. 3c). ZjRNase1has two introns, while ZjRNase3 has one intron. ZjRNase2/4 has more than 2 introns (Fig. 3c). MEME analysis was performed online to identify additional motifs among the 4 ZjRNases. Five conserved motifs were predicted (Fig. 3b), and each ZjRNase contained four or five of them. Several motifs were common to most members. Compared to the other three members, ZjRNase1 lacked motif 4 (light blue box in Fig. 3b).

Fig. 3
figure 3

Phylogenetic relationships, gene structure and gene architecture of the conserved protein motifs in ZjRNases. a Phylogenetic tree was constructed based on the full-length sequences of ZjRNase proteins. b Motif composition. The motifs, numbered 1–5, were displayed in different coloured boxes. c Exon–intron structure of ZjRNases. The light green boxes indicate untranslated 5′- and 3′-regions; the yellow boxes indicate exons; and the black lines indicate introns

Full size image

Analysis of cis-acting elements in the promoter region of ZjRNases

The cis-acting elements were found to be related to hormone responses, development, light responses, promoter site binding and other functions (Fig. 4). The important elements are light-responsive elements, including Box 4, G-Box, TCT motif, CAAT-box, GATA motif and GATA motif elements. Six hormone-responsive elements were identified, and the jujube T2 family members were found to contain 6 to 12 light-responsive elements.

Fig. 4
figure 4

Prediction of cis-acting elements in ZjRNases. a Numbers of cis-acting elements detected in the promoter region of each ZjRNase gene. All the cis-acting elements were divided into six types

Full size image

Analysis of protein structure and protein interactions

The results of protein structure prediction analysis showed that ZjRNase1 is similar to ZjRNase3 and that ZjRNase2 is similar to ZjRNase4. These results are consistent with those of the above-mentioned gene structural analysis (Fig. 5a). Protein interaction prediction analysis predicted only AtRNS1 (ZjRNase1), AtRNS2 (ZjRNase2), and AtRNS3 (ZjRNase3). AtRNS4 (ZjRNase4) had no prediction results (Fig. 5b). Many proteins interacted with ZjRNase proteins, some of which were transcription factors, such as NAC (NAC genes) and EIN2 (INSENSITIVE 2).

Fig. 5
figure 5

ZjRNase gene family protein analysis. a Protein structure analysis of 4 ZjRNases via the Phyre2 database. b Protein–protein interaction analysis of 3 ZjRNases by the STRING database

Full size image

The transcriptome sequencing analysis of jujube fruits

Further through the analysis of the published winter jujube transcriptome data showed that ZjRNase1 and ZjRNase2 were expressed in jujube fruits, but hardly ZjRNase3 and ZjRNase4 (Fig. 6). The results indicate that ZjRNase1 and ZjRNase2 may be involved in fruit and seed development. In order to further understand the function of ZjRNase1 and ZjRNase2, subsequent transgenic experiments were carried out.

Fig. 6
figure 6

The leaves morphology of the ZjRNase1 three representative transgenic lines. F1 represents the young fruit period, F2 represents the bulge period fruit, F3 represents the white-ripe period fruit, F4 represents the half-red period fruit, and F5 represents the whole-red period fruit

Full size image

Overexpression of ZjRNase1 and ZjRNase2 in Arabidopsis thaliana

Recombinant vectors overexpressing ZjRNase1 and ZjRNase2 were constructed. Arabidopsis thaliana (Col-0) plants were transformed via Agrobacterium-mediated infection, and the seeds were collected. Transgenic plants with ZjRNase1 and ZjRNase2 were obtained by screening resistant seedlings via hygromycin-containing media. The leaves of the transgenic lines overexpressing ZjRNase1 were significantly curled and twisted (Fig. 7). Further observations under an anatomical microscope of the transgenic lines overexpressing ZjRNase2 revealed that the siliques were crisp, with small trichomes around them (Fig. 8).

Fig. 7
figure 7

The leaves morphology of the ZjRNase1 three representative transgenic lines (#OE1, #OE2 and #OE3) and WT. Scale bar.1 cm a Growth morphology of three representative ZjRNase1 transgenic lines and WT after planted for 7 days. Scale bar.1 cm b The frontal morphology of rosette leaves of three representative ZjRNase1 transgenic lines and WT after planted for 14 days. Scale bar.1 cm c The abaxial morphology of rosette leaves of three representative ZjRNase1 transgenic lines and WT after planted for 14 days. Scale bar.1 cm d The frontal morphology of the inflorescence stem leaf of three representative ZjRNase1 transgenic line and the WT after planted on the 21 days. Scale bar.1 cm e The abaxial morphology of the inflorescence stem leaf of three representative ZjRNase1 transgenic line and the WT planted on the 21d. Scale bar.1 cm, WT: Arabidopsis thaliana (Col-0), OE: overexpressionZjRNase1

Full size image
Fig. 8
figure 8

Morphology of siliques of transgenic ZjRNase2 overexpression plants. Scale bar: 1 cm

Full size image

The siliques of the ZjRNase overexpression plants were significantly shorter than those of the Arabidopsis thaliana (Col-0) plants (Fig. 9). Tissue-specific expression of the leaves, stems, flowers and siliques of the overexpression plants revealed that ZjRNase1 was mainly expressed in the siliques, but ZjRNase2 was mainly expressed in the flowers (Fig. 10).

Fig. 9
figure 9

Morphology of siliques of the three representative transgenic ZjRNase1and ZjRNase2overexpression lines(#OE1,#OE2 and #OE3) and WT plants. Scale bar: 1 cm. WT: Arabidopsis thaliana (Col-0), OE: overexpression ZjRNase1 and ZjRNase2

Full size image
Fig. 10
figure 10

qRT-PCR detection of transgenic plants. a. Tissue-specific expression of the ZjRNase1 and ZjRNase2. b. Tissue expression of the ZjRNase1 ZjRNase2 three represenative transgenic lines (#OE1, #OE2 and #OE3) and WT. WT: Arabidopsis thaliana (Col-0), OE: overexpression ZjRNase1 and ZjRNase2

Full size image

The seeds were harvested from the T1 generation of ZjRNase1 and ZjRNase2 overexpression transgenic lines. The seeds were screened via media containing hygromycin, and the phenotype of the T3-generation overexpression plants was the same as that of the T2- and T1-generation plants.

Through statistical analysis of the siliques and seeds of the ZjRNase1 and ZjRNase2 overexpression plants, the siliques of the overexpression plants were significantly shorter than those of the Arabidopsis thaliana (Col-0), with the ZjRNase2 overexpression plants presenting the most significant results, only 4.80 mm long (Fig. 11). In addition, the podetium length of the ZjRNase2 overexpression plants was significantly longer than that of the Arabidopsis thaliana (Col-0). Compared with that of the Arabidopsis thaliana (Col-0), the number of seeds produced by the ZjRNase1 and ZjRNase2 overexpression plants was significantly reduced. The average seed number of the ZjRNase1 overexpression plants was only approximately 50% that of the Arabidopsis thaliana (Col-0). Interestingly, the seed number significantly decreased for ZjRNase2 overexpression lines 1, 2, and 3 to approximately 50, 8.33, and 0% of that for the Arabidopsis thaliana (Col-0), so seed production was absent in the ZjRNase2 overexpression transgenic line 3 (Figs. 11 and 12). The results showed that overexpression of the ZjRNase1 and ZjRNase2 genes in Arabidopsis can cause reduced seed content. Additionally, this overexpression can cause morphological changes in other tissues. The leaves of the ZjRNase1 overexpression transgenic lines were curled and twisted. Moreover, the siliques of the ZjRNase2 overexpression lines were wrinkled with few or even no seeds.

Fig. 11
figure 11

Seeds in the siliques of three representative transgenic lines (#OE1,#OE2 and #OE3) and WT. Scale bar: 1 mm, WT: Arabidopsis thaliana (Col-0), OE: overexpressionZjRNase1and ZjRNase2

Full size image
Fig. 12
figure 12

Statistical analysis of the siliques, carpopodium and seeds of three representative transgenic lines (#OE1,#OE2 and #OE3) and WT. The error bars represent the ±SDs of three independent replicates (***, p < 0.001;**, p < 0.01; *, p < 0.05 Duncan’s multiple range test).WT: Arabidopsis thaliana (Col-0), OE: overexpressionZjRNase1, ZjRNase2

Full size image

Discussion

Ribonuclease (RNase T2) play crucial roles in plant evolution and breeding and an increasing number of RNase T2/S-RNase enzymes have subsequently been reported in genome of different plants, such as Acacia, Solanaceae and Rosaceae [3, 8,9,10]. Through genome-wide mining, four members of the RNase T2 family were identified in jujube. These members could be divided into two categories according to phylogenetic analysis, which is relatively low compared with that of other species [1, 5].

The previous study showed that RNase T2 is related to self-incompatibility and abiotic stress responses in plants [1, 3, 5]. Some genes involved in seed and fruit development have been reported [21, 22]. For instance, YABBY、OVATE and EPFL2 influenced fruit size and shape, ORANGE and MPK4 were related to seed number [23,24,25,26,27,28,29]. However, the research about RNase T2 function during fruit and seed development was absent. In terms of bioinformatics, the RNase T2 gene family members in jujube have characteristics similar to those of RNases of other plant species [1, 5]. In particular, the jujube RNase T2 gene retains the original ability of T2 genes, and when overexpressed in Arabidopsis, these genes could lead to reduced seed production. The VvNAC26 transgenic plants were found to reduce tomato seeds [30] and INSENSITIVE2 (EIN2) encodes a membrane protein and affected seed development [31]. This study showed that ZjRNase1 and ZjRNase2 had interaction with NAC and EIN2, respectively. It indicated NAC and EIN2 transcription factors were probably associated with function of ZjRNase1 and ZjRNase2 involved in seed development.

In addition, the jujube RNase T2 family members showed some differences in terms of their function. The class I member ZjRNase1, after being overexpressed, was found to be highly expressed only in flowers and caused a twisted-leaf phenotype in addition to siliques shorten and reduced seed number. Analysis of its promoter revealed that the sequence (CAAT (A/T) ATTG) may participate in the differentiation of the palisade tissue and can also bind HD-ZIPI transcription factors, which have been shown to play a role in leaf morphogenesis [32]. In addition, HD-ZIPI proteins were identified as capable of interacting with RTNLB8 and VAP27-1 in terms of protein interactions [33]. All of these interacting proteins are highly expressed during leaf morphological development. The class II member ZjRNase2 was expressed in the flowers of the transgenic lines. Overexpression of this gene caused crisp siliques, reduced seed numbers and even no seeds, as well as the production of trichomes. According to the literature, plants produce more trichomes to resist pests, indicating that such gene may also be associated with insect resistance [34, 35].

Conclusions

In this study, four ZjRNase genes and their corresponding protein sequences were identified from the jujube genome, and the ZjRNase genes were divided into two categories. Class I had member ZjRNase1 and ZjRNase2, ZjRNase3 and ZjRNase4 belonged to Class II but not expressed in the fruit. Therefore, ZjRNase1 and ZjRNase2 were selected for functional verification. As a result of the transgene, we founded that ZjRNase1 and ZjRNase2 could lead to a decrease in the number of seeds, inferring that ZjRNase genes might be involved in the formation of seeds. This study provides a basis for further studies on the functional properties of RNase genes; however, further studies are needed to better elucidate the regulatory mechanism of these four RNase genes on seed formation in jujube breeding.

Materials and methods

Plant materials and cultivation

Arabidopsis thaliana (Col-0) seeds were obtained from Xuan Zhao from China Agriculture University and sown in a soil medium matrix (peat: vermiculite = 1:1) under a 16 h light/8 h darkness photoperiod at 20 ± 2 °C and a relative humidity of 60 ± 5%. The seeds of Arabidopsis thaliana (Col-0) plants were grown on 1/2-strength Murashige and Skoog (MS) media. All the plants were grown under the same conditions. RNA was extracted using a TIANGEN plant RNAprep Pure Plant Kit DP432 (TIANGEN biotech company). Jujube fruit transcriptome data were obtained from the National Center for Biotechnology Information (NCBI) database [19].

Database searches and identification of RNase genes in the Ziziphus jujuba genome

The genome sequences of Ziziphus jujuba Mill. were obtained from the National Center for Biotechnology Information (NCBI) database [19]. The sequences of 14, 9 identified MdRNases, PyRNases were downloaded from the National Center for Biotechnology Information (NCBI) database. In addition, the sequences of8 identified OsRNases were downloaded [1]. RNases were identified by two rounds of BLASTP searches. First, the sequences of all OsRNases were used to search for possible ZjRNases sequences via TBtools [36]. Then, NCBI Batch CD-Search was used to confirm whether the candidate RNases contained the RNase_T2 superfamily domain (pfam00445) or the Ribonuclease_T2 domain (cl00208). A total of 4 ZjRNase genes were ultimately identified in the genome. The protein length, isoelectric point (pI) and molecular weight (MW) were subsequently predicted.

Phylogenetic analysis and multiple sequence alignment

Sequences of the OsRNase proteins were obtained from the Phytozome database. A neighbour-joining (NJ) phylogenetic tree comprising the full-length sequences of MdRNases, PyRNases and ZjRNases was constructed with 1000 bootstrap replicates using MEGA 7.0. Multiple sequence alignment of all ZjRNases was also performed by MEGA 7.0.

RNase gene structure and conserved motif analysis

The RNase gene structure and conserved domains were analysed and visualized using TBtools software [36]. The conserved motifs of the identified ZjRNase proteins were explored with the help of the Multiple Expectation Maximization for Motif Elicitation (MEME) online program.

Analysis of cis-acting elements of ZjRNases

The potential regulatory cis-acting elements of jujube RNases were checked by using TBtools software; the region 2000 bp upstream of the start codon was evaluated. Then, the cis-acting elements in the promoter were predicted via PlantCARE online software to identify their regulatory functions.

Protein structure and protein–protein interaction predictions

The amino acid sequences of 4 ZjRNases were submitted to Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/) for protein structure analysis [37]. Similarly, the amino acid sequences of the same 4 ZjRNases were submitted to the STRING database (https://string-db.org/) for protein–protein interaction analysis. The orthologues of these genes in Arabidopsis thaliana were selected as references.

RNA extraction and quantitative real-time PCR (qPCR) analysis

The extraction of total RNA from leaves and subsequent cDNA synthesis were performed as described previously [38]. Gene expression was then analyzed via qPCR [39]. We included at least three independent biological replicates and three technical replicates. First-strand cDNA was synthesized from RNA with a PrimeScript RT Reagent Kit (TIANGEN). qPCR was carried out on 20 μL reaction mixtures by the use of SYBR Green fluorescence (TransGen Biotech, China) in conjunction with a Roche LightCycler® 480 Real-Time PCR System [40]. The AtActin2 was used as the internal reference control [39]. Relative gene expression levels were calculated according to the 2-ΔΔCT method [41]. All the primers used for qPCR are listed in the Additional file 1.

Plasmid construction and gene overexpression

According to the bioinformatics analysis above, ZjRNase1 and ZjRNase2 were selected for gene functional analysis. Plasmid construction and agroinfiltration assays of Arabidopsis thaliana (Col-0) were performed based on the sequences above. We cloned the coding DNA sequences (CDS) of ZjRNase1 and ZjRNase2and inserted them into a stable overexpression vector (PBI121) [42]. All the primers used for qPCR are listed in the Additional file 1. The above-mentioned vector construct was infected into Arabidopsis thaliana (Col-0) through the transformation method mediated by Agrobacterium (GV3101 strain) and infection buffer (5% sucrose, 0.04% Silwet-77). Infected Arabidopsis thaliana (Col-0) plants was incubated in the dark for 24 h.

Seed observation experiment

The ripe siliques were washed with distilled water, placed in a 95% ethanol and acetic acid (3:1) fixative solution for 24 hours, and then transferred to 70% ethanol and stored in a refrigerator at 4 °C overnight. The next day, it was transferred into 85 and 95% ethanol, dehydrated for 1 h respectively, and dehydrated in absolute ethanol 3 times, the first two times were 1 h, and the last time was 5 h. soak with ethanol and methyl salicylate (1:1) for 1 h, and soak with pure methyl salicylate for 3 times, the first 2 times for 1 h, and the last time for 24 h. Then observed under the microscope and took pictures for preservation.

Statistical analysis

The experiments were performed for three technical replications. One-way analysis of variance (ANOVA) was used to determine the statistical significance at p ≤ 0.05.Statistically signifcant diferences were indicated either with * (P < 0.05), ** (P < 0.01), *** (P < 0.001).

Availability of data and materials

The entire Ziziphus jujuba Mill. genome sequence information was obtained from the Ensembl Genomes website (https://www.ncbi.nlm.nih.gov/assembly/GCF_000826755.1). Arabidopsis thaliana (Col-0) materials used in the experiment were supplied by Doctor. Xuan Zhao of Hebei Agricultural University, and this material was used with permission. The datasets supporting the conclusions of this study are included in the article and its additional files.

Abbreviations

WT:

Arabidopsis thaliana (Col-0)

OE:

Overexpression ZjRNase1, ZjRNase2

qRT-PCR:

Quantitative real-time PCR

MW:

Molecular weight

PI:

Theoretical isoelectric point

References

  1. Macintosh G, Hillwig M, Meyer A, FlagelL. Rnase t2 genes from rice and the evolution of secretory ribonucleases in plants. Mol Gen Genomics. 2010;283:381–96. https://doi.org/10.1007/s00438-010-0524-9.

    Article  CAS  Google Scholar 

  2. Sato K, Egami F. Studies on ribonucleases in takadiastase I. J Biochem. 1957;44:753–67. https://doi.org/10.1093/oxfordjournals.jbchem.a126717.

    Article  CAS  Google Scholar 

  3. McClure B, Haring V. Style self-incompatibility gene products of nicotiana alata are ribonucleases. Nature. 1989;342:955–7. https://doi.org/10.1038/342955a0.

    Article  CAS  PubMed  Google Scholar 

  4. Kawata Y, Sakiyama F, Tamaoki H. Amino-acid sequence of ribonuclease T2 from Aspergillus oryzae. Eur J Biochem. 1998;176:683–97. https://doi.org/10.1111/j.1432-1033.1988.tb14331.x.

    Article  Google Scholar 

  5. Liang M, Yang W, Su S, Fu L, Yi H, Chen C. Genome-wide identification and functional analysis of S-RNase involved in the self-incompatibility of citrus. Mol Gen Genomics. 2017;292:325–41. https://doi.org/10.1007/s00438-016-1279-8.

    Article  CAS  Google Scholar 

  6. Qin M, Zhang Y, Liu J, Li M. Molecular mechanisms underlying the participation of ribonuclease t2 gene into self-incompatibility of citrus grandis var. shatianyu hort. Cell Mol Biol. 2018;64:1153. https://doi.org/10.14715/cmb/2018.64.2.22.

    Article  PubMed  Google Scholar 

  7. Deshpande R, Shankar V. Ribonucleases from T2 family. Crit Rev Microbiol. 2002;28:79–122. https://doi.org/10.1080/1040-840291046704.

    Article  CAS  PubMed  Google Scholar 

  8. Xue Y, Carpenter R, Coen D. Origin of allelic diversity in antirrhinum s locus rnases. Plant Cell. 1996;8:805–14. https://doi.org/10.1105/tpc.8.5.805.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Ramanauskas K. The evolutionary history of plant t2/s-type ribonucleases. Peerj. 2017;5(2167):8359. https://doi.org/10.7717/peerj.3790.

    Article  CAS  Google Scholar 

  10. Zhao H, Zhang Y, Zhang H, Song Y, Zhao F, Zhang Y. Origin, loss, and regain of self-incompatibility in angiosperms. Plant Cell. 2021;34:579–96. https://doi.org/10.1093/plcell/koab266.

    Article  PubMed Central  Google Scholar 

  11. Sassa H, Hirano H, Ikehashi H. Identification and characterization of stylar glycoproteins associated with self-incompatibility genes of japanese pear. pyrus serotina rehd. Molgengenet. 1993;241:17–25. https://doi.org/10.1007/BF00280196.

    Article  CAS  Google Scholar 

  12. Burgos L, Pereztornero O, Ballester J, Olmos E. Detection and inheritance of stylar ribonucleases associated with incompatibility alleles in apricot. Sex Plant Reprod. 1998;11:153–8. https://doi.org/10.1007/s004970050133.

    Article  CAS  Google Scholar 

  13. Wang S, Qian W, Ying Z, Qie H, Wang H. Identification of two new S-RNases and molecular s-genotyping of twenty loquat cutivars [eriobotrya japonica (thunb.) lindl.]. Sci Hortic. 2017;218:48–55. https://doi.org/10.1016/j.scienta.2017.02.002.

    Article  CAS  Google Scholar 

  14. Gho Y, Choi H, Moon S, Song M, Park H, Kim D, et al. Phosphate-starvation-inducible S-like RNase genes in Rice are involved in phosphate source recycling by RNA decay. Frontiers. Plant Sci. 2020;11:585561. https://doi.org/10.3389/fpls.2020.585561.

    Article  Google Scholar 

  15. Qu Z, Wang Y. Chinese fruit trees record·Chinese jujube. Beijing: Chinese Forestry Publisher Press; 1993. p. 5–6, 45, 65.

    Google Scholar 

  16. Huang J, Zhang C, Xing Z, Fei Z, Wan K, Zhong Z. The jujube genome provides insights into genome evolution and the domestication of sweetness/acidity taste in fruit trees. PLoS Genet. 2016;12:12. https://doi.org/10.1371/journal.pgen.1006433.

    Article  CAS  Google Scholar 

  17. Liu M, Wang J. Fruit scientific research in new China in the past 70 years: Chinese jujube. Journal of fruit. Science. 2019;36:1369–81. https://doi.org/10.13925/j.cnki.gsxb.Z11.

    Article  Google Scholar 

  18. Liu M, Wang J, Wang L, Liu P, Zhao J, Zhao Z, et al. The historical and current research progress on jujube-a superfruit for the future. Horticult Res. 2020;7:119. https://doi.org/10.1038/s41438-020-00346-5.

    Article  CAS  Google Scholar 

  19. Liu M, Zhao J, Cai Q, Liu G, Wang J, Zhao Z. The complex jujube genome provides insights into fruit tree biology. Nat Commun. 2014;5:5315. https://doi.org/10.1038/ncomms6315.

    Article  CAS  PubMed  Google Scholar 

  20. Irie M. Structure-function relationships of acid ribonucleases: lysosomal, vacuolar, and periplasmic enzymes. Pharmacol Ther. 1999;81:77–89. https://doi.org/10.1016/s0163-7258(98)00035-7.

    Article  CAS  PubMed  Google Scholar 

  21. Barbera G, Inglese P. Seed content and fruit characteristics in cactus pear (opuntia ficus-indica mill.). Sci Horticult. 1994;58:161–5. https://doi.org/10.1016/0304-4238(94)90136-8.

    Article  Google Scholar 

  22. Zhu Y, Ye J, Zhan J, Zheng X, Wang H. Validation and characterization of a seed number per silique quantitative trait locus qsn.a7 in rapeseed (brassica napus l.). Frontiers. Plant Sci. 2020;11:68. https://doi.org/10.3389/fpls.2020.00068.

    Article  Google Scholar 

  23. Kawamoto N, Carpio D, Hofmann A, MizutaY SR. Peptide pair coordinates regular ovule initiation patterns with seed number and fruit size. Curr Biol. 2019;16:4352–61. https://doi.org/10.1016/j.cub.2020.08.050.

    Article  CAS  Google Scholar 

  24. Xiao W, Hu S, Zou X, Cai R, Liao R, Lin X. Lectin receptor-like kinase lecrk-viii.2 is a missing link in mapk signaling-mediated yield control. Plant Physiol. 2021;187:303–20. https://doi.org/10.1093/plphys/kiab241.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Yazdani M, Sun Z, Yuan H, Zeng S, Thannhauser T, Vrebalov J, et al. Ectopic expression of ORANGE promotes carotenoid accumulation and fruit development in tomato. Plant Biotechnol J. 2019;17(1):33–49. https://doi.org/10.1111/pbi.12945.

    Article  CAS  PubMed  Google Scholar 

  26. Wang J, Li M, Zhuo S, Liu Y, Yu X, Mukhtar S, et al. Mitogen-activated protein kinase 4 is obligatory for late pollen and early fruit development in tomato. Hortic Res. 2022;9:uhac048. https://doi.org/10.1093/hr/uhac048.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Kawamoto N, Del Carpio D, Hofmann A, Mizuta Y, Kurihara D, Higashiyama T, et al. A peptide pair coordinates regular ovule initiation patterns with seed number and fruit size. Curr Biol. 2020;30(22):4352–4361.e4. https://doi.org/10.1016/j.cub.2020.08.050.

    Article  CAS  PubMed  Google Scholar 

  28. Snouffer A, Kraus C, Vander K. The shape of things to come: ovate family proteins regulate plant organ shape. Curr Opin Plant Biol. 2020;53:98–105. https://doi.org/10.1016/j.pbi.2019.10.005.2019.

    Article  CAS  PubMed  Google Scholar 

  29. Rodríguez G, Muños S, Anderson C, Sim S, Michel A, Causse M, et al. Distribution of SUN, OVATE, LC, and FAS in the tomato germplasm and the relationship to fruit shape diversity. Plant Physiol. 2011;156(1):275–85. https://doi.org/10.1104/pp.110.167577.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Zhang S, Dong R, Wang Y, Li X, Ji M, Wang X. NAC domain gene VvNAC26 interacts with VvMADS9 and influences seed and fruit development. Plant Physiol Biochem. 2021;164:63–72. https://doi.org/10.1016/j.plaphy.

    Article  CAS  PubMed  Google Scholar 

  31. Atsumi A, Ryan C, Hong Q, Jeffrey C. Endosperm and maternal-specific expression of EIN2 in the endosperm affects endosperm cellularization and seed size in Arabidopsis. Genetics. 2022:iyac161. https://doi.org/10.1093/genetics/iyac161.

  32. Franco J, Lopez V, Carrasco J, Godoy M, Vera P, Solano R. Dna-binding specificities of plant transcription factors and their potential to define target genes. Proc Natl Acad Sci U S A. 2014;111:2367–72. https://doi.org/10.1073/pnas.1316278111.

    Article  CAS  Google Scholar 

  33. Klepikova A, Kasianov A, Gerasimov E, Logacheva M, Penin A. A high resolution map of the arabidopsis thaliana developmental transcriptome based on rna-seq profiling. Plant J. 2016;88:1058–70. https://doi.org/10.1111/tpj.13312.

    Article  CAS  PubMed  Google Scholar 

  34. Ma W, Lu M, Ludlow R, Wang D, Zeng J, An H. Contrastive analysis of trichome distribution, morphology, structure, and associated gene expression reveals the formation factors of different trichome types in two commercial Rosa species. Sci Hortic. 2021;285:110–31. https://doi.org/10.1016/j.scienta.2021.110131.

    Article  CAS  Google Scholar 

  35. ChenC LM, Jiang L, Liu X, Zhao J, Yan S. Transcriptome profiling reveals roles of meristem regulators and polarity genes during fruit trichome development in cucumber (Cucumis sativus l.). J Exp Bot. 2014;17:4943–58. https://doi.org/10.1093/jxb/eru258.

    Article  CAS  Google Scholar 

  36. Chen C, Chen H, Zhang Y, Thomas H, Xia R. TBtools: An integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020;13:1194–202. https://doi.org/10.1016/j.molp.2020.06.009.

    Article  CAS  PubMed  Google Scholar 

  37. Alexey D, Christian C, James P, Barton G. Jpred4: a protein secondary structure prediction server. Nucleic Acids Res. 2015;43:W389–94. https://doi.org/10.1093/nar/gkv332.

    Article  CAS  Google Scholar 

  38. Liu Z, Zhang L, Xue C, Fang H, Zhao J, Liu M. Genome-wide identification and analysis of MAPK and MAPKK gene family in Chinese jujube (Ziziphus jujuba mill.). BMC Genomics. 2017;18:855. https://doi.org/10.1186/s12864-017-4259-4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Stranne M, Ren Y, Fimognari L, Birdseye D, SakuragiY. Tbl10 is required for o -acetylation of pectic rhamnogalacturonan-i in arabidopsis thaliana. Plant J. 2018;96(4):772–85. https://doi.org/10.1111/tpj.14067.

    Article  CAS  PubMed  Google Scholar 

  40. Wang L, Li M, Liu Z, Dai L, Zhang M, Wang L. Genome-wide identification of cngc genes in chinese jujube (ziziphus jujuba mill.) and zjcngc2 mediated signalling cascades in response to cold stress. BMC Genomics. 2020;21:191. https://doi.org/10.1186/s12864-020-6601-5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Livak K, Schmittgen T. Analysis of relative gene expression data using real-time quantitative PCR and the 2-[Delta][Delta] CT method. Methods. 2001;25:402–8. https://doi.org/10.1006/meth.2001.1262.

    Article  CAS  PubMed  Google Scholar 

  42. Jefferson R. Gus fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 1987;6:3901–7. https://doi.org/10.1002/j.1460-2075.1987.tb02730.x.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank all of the colleagues in our laboratory for providing useful discussions and technical assistance. We are very grateful to the editor and reviewers for critically evaluating the manuscript and providing constructive comments for its improvement.

Funding

This work was supported by grants from the National Key R&D Program Project Funding (2020YFD1000705, 2019YFD1001605), National Natural Science Foundation (32101542), Hebei Province Key R&D Program (20326811D, 21326304D), The Science and Technology Research Project of University in Hebei Province (QN2020205).

Author information

Author notes

  1. Zhi Luo, Yu Zhang and Chunjiao Tian contributed equally to this work.

Authors and Affiliations

  1. College of Horticulture, Hebei Agricultural University, Baoding, 071001, China

    Zhi Luo, Xuan Zhao, Zhiguo Liu, Lili Wang, Lixin Wang & Mengjun Liu

  2. Research Center of Chinese Jujube, College of Horticulture, Hebei Agricultural University, Baoding, 071001, China

    Zhi Luo, Xuan Zhao, Zhiguo Liu, Lili Wang, Lixin Wang & Mengjun Liu

  3. College of Forestry, Hebei Agricultural University, Baoding, 071001, China

    Yu Zhang, Chunjiao Tian & Jiurui Wang

  4. School of Landscape and Ecological Engineering, Hebei University of Engineering, Handan, 056038, China

    Lihu Wang

  5. College of Life Science, Hebei Agricultural University, Baoding, 071001, China

    Jin Zhao

Authors
  1. Zhi Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yu Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chunjiao Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lihu Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xuan Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zhiguo Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lili Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Lixin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jin Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jiurui Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Mengjun Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Zhi Luo, Yu Zhang and Chun-Jiao Tian: materials preparation, performing experiment, data analysis and wrote the main manuscript text. Li-Hu Wang, Zhi-Guo Liu, Li-Li Wang, Xuan Zhao and Li-Xin Wang:experimental-guidance and data analysis. Jiu-Rui Wang: experimental design, original draft, revision and final draft. Meng-Jun Liu,Jiu-Rui Wang and Jin Zhao: experimental-guidance, revision and final draft. All authors reviewed and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Jin Zhao, Jiurui Wang or Mengjun Liu.

Ethics declarations

Ethics approval and consent to participate

This article does not contain any studies with human participants or animals performed by the authors. These methods were carried out in accordance with relevant guidelines and regulations including the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. Arabidopsis thaliana (Col-0) materials used in the experiment were supplied by Doctor. Xuan Zhao of Hebei Agricultural University, and this material was used with permission. All experimental protocols were approved by Hebei Agricultural University.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1.

The primers used for qRT-PCR and plasmid construction.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Luo, Z., Zhang, Y., Tian, C. et al. Genome-wide screening of the RNase T2 gene family and functional analyses in jujube (Ziziphus jujuba Mill.). BMC Genomics 24, 80 (2023). https://doi.org/10.1186/s12864-023-09165-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12864-023-09165-z

Keywords

  • Ziziphus jujuba Mill.
  • RNase T2
  • ZjRNase1
  • ZjRNase2
  • Seed

BMC Genomics

ISSN: 1471-2164

Contact us