Ras-Related Nuclear Protein Ran3B Gene Is Involved in Hormone Responses in the Embryogenic Callus of Dimocarpus longan Lour.

Ras-related guanosine triphosphate (GTP)-binding nuclear protein (Ran) GTPases function as molecular switches and regulate diverse cellular events in eukaryotes. Our previous work suggested that DlRan3B is active during longan (Dimocarpus longan Lour.) somatic embryogenesis (SE) processes. Herein, subcellular localization of DlRan3B was found to be localized in the nucleus and expression profiling of DlRan3B was performed during longan SE and after exposure to plant hormones (indoleacetic acid (IAA), gibberellin A3 (GA3), salicylic acid (SA), methyl jasmonte (MeJA), and abscisic acid (ABA)). We cloned and sequenced 1569 bp of 5′-flanking sequence of DlRan3B (GenBank: JQ279697). Bioinformatic analysis indicated that the promoter contained plant hormone-related regulatory elements. Deletion analysis and responses to hormones identified stimulative and repressive regulatory elements in the DlRan3B promoter. The key elements included those responding to auxin, gibberellin, SA, MeJA, and ABA. DlRan3B was located in the nucleus and accumulated in the late stage of longan SE. The expression of DlRan3B was significantly induced by IAA, GA3, and ABA, but suppressed by SA and MeJA. Promoter transcription was induced by IAA and GA3, but suppressed by SA. Thus, DlRan3B might participate in auxin, gibberellin, and ABA responses during longan late SE, and DlRan3B is involved in phytohormone responsiveness.


Introduction
Ran GTPases regulate a multiple series of cell activities by functioning as molecular switches in animals [1][2][3]. In plants, Ran GTPases are involved in various responses, including mediating hormone sensitivities [4][5][6]. Furthermore, overexpression of a Ran GTPase homolog in different plants has caused a variety of developmental abnormalities, such as increases in primordial tissue, mitotic index, sensitivity to exogenous auxin and ABA treatments, and decreases in lateral root number [7][8][9][10]. Meanwhile, the mutations in some Ran GTPase homologs can lead to male sterility [9]. These results have led to a conclusion that Ran is essential for plant development and also serve as clues to the potential function of Ran in hormone signaling transduction in plants.
Longan tree (Dimocarpus longan Lour.) is one of the evergreen fruit trees grown in southern China and its fruit have important applications in food industry and health care [11][12][13]. The development of longan seeds is crucial for longan fruit development, including fruit appearance and processes of fruit setting and ripening. Plant Ran might be involved in the cell activities during plant embryos development, because of its animal its counterparts' tissue-specific expression during embryogenesis and its role in cell division in animal embryos [14][15][16]. Nevertheless, characterization of plant Ran, especially its involvement in plant embryogenesis and hormone transduction, remains poorly reported. 2 of 13 In previous studies, full-length cDNAs and DNAs of DlRan3 were cloned from longan somatic embryos and thereafter two 5 1 flanking sequences (1256 and 714 bp) of DlRan3A (GenBank: JQ775539) and DlRan3B (GenBank: JQ279697), respectively, were isolated. It was analyzed by bioinformatics that major elements in the promoters were closely related to phytohormones [17]. A previous study also showed DlRan3A's possible participation in auxin signaling transduction in early stages of somatic embryogenesis (SE) in longan and its possible roles in plant hormone, light, and abiotic stress responsiveness. However, to date, little is known about how DlRan3B functions in plant hormone responsiveness in plant embryos.
To further reveal the biological role of DlRan3B, its protein subcellular location was observed and its expression profile was analyzed during longan SE processes and under treatments of exogenous plant hormone, including indoleacetic acid (IAA), gibberellin A3 (GA 3 ), salicylic acid (SA), methyl jasmonte (MeJA), and abscisic acid (ABA). Furthermore, to understand the regulatory role of DlRan3B, its 5 1 flanking sequence was isolated and characterized to identify an efficient promoter that could initiate the constitutive expression of a foreign gene in transgenic plants. Furthermore, deletion analysis and different transcriptional activities in response to phytohormones showed that the DlRan3B promoter has positive and negative regulatory elements. This study provides a multifaceted view of the potential roles of DlRan3B during longan somatic embryo formation and in phytohormone signaling pathways.

Subcellular Localization of DlRan3B
A fusion protein of DlRan3B-mGFP (green fluorescent protein) was transiently expressed in the epidermal cells of tobacco leaves to detect subcellular localization of DlRan3B. As a result, DlRan3B-mGFP was dominantly located in the nucleus (Figure 1 and Figure S1). In previous studies, full-length cDNAs and DNAs of DlRan3 were cloned from longan somatic embryos and thereafter two 5′ flanking sequences (1256 and 714 bp) of DlRan3A (GenBank: JQ775539) and DlRan3B (GenBank: JQ279697), respectively, were isolated. It was analyzed by bioinformatics that major elements in the promoters were closely related to phytohormones [17]. A previous study also showed DlRan3A's possible participation in auxin signaling transduction in early stages of somatic embryogenesis (SE) in longan and its possible roles in plant hormone, light, and abiotic stress responsiveness. However, to date, little is known about how DlRan3B functions in plant hormone responsiveness in plant embryos.
To further reveal the biological role of DlRan3B, its protein subcellular location was observed and its expression profile was analyzed during longan SE processes and under treatments of exogenous plant hormone, including indoleacetic acid (IAA), gibberellin A3 (GA3), salicylic acid (SA), methyl jasmonte (MeJA), and abscisic acid (ABA). Furthermore, to understand the regulatory role of DlRan3B, its 5′ flanking sequence was isolated and characterized to identify an efficient promoter that could initiate the constitutive expression of a foreign gene in transgenic plants. Furthermore, deletion analysis and different transcriptional activities in response to phytohormones showed that the DlRan3B promoter has positive and negative regulatory elements. This study provides a multifaceted view of the potential roles of DlRan3B during longan somatic embryo formation and in phytohormone signaling pathways.

Subcellular Localization of DlRan3B
A fusion protein of DlRan3B-mGFP (green fluorescent protein) was transiently expressed in the epidermal cells of tobacco leaves to detect subcellular localization of DlRan3B. As a result, DlRan3B-mGFP was dominantly located in the nucleus (Figure 1 and Figure S1).

The Expression Profiling of DlRan3B During Longan Somatic Embryogenesis (SE)
To discover the transcriptional control of DlRan3B during longan SE, we measured the transcript levels of DlRan3B during longan SE. DlRan3B showed decreasing expression during primary developmental stages, with the least DlRan3B accumulation observed in the globular

The Expression Profiling of DlRan3B During Longan Somatic Embryogenesis (SE)
To discover the transcriptional control of DlRan3B during longan SE, we measured the transcript levels of DlRan3B during longan SE. DlRan3B showed decreasing expression during primary developmental stages, with the least DlRan3B accumulation observed in the globular embryo (GE) stage, while increasing expression was observed during the middle and late stages of SE, with the highest DlRan3B accumulation in the cotyledon embryo (CE) stage. The distinctive pattern suggested an important role of DlRan3B accumulation during longan late SE, rather than early stages ( Figure 2).  Figure 2).

The Effect of Exogenous Plant Hormones on DlRan3B Expression
To understand how DlRan3B responds to plant hormones, we analyzed the relative levels of DlRan3B mRNA in longan embryogenic calluses (ECs) treated with different concentrations of IAA, GA3, SA, MeJA, and ABA ( Figure 3A-E). Among the treatments, 26.0 μM GA3 enhanced the DlRan3B transcript level to approximately 1.6-fold in contrast to the control ( Figure 3B); ABA induced a slight, positive transcriptional control on DlRan3B, with no dose-dependent effects ( Figure 3E). IAA over the range of 2.9-8.6 μM increased DlRan3B expression, with 8.6 μM IAA inducing a 1.3-fold level to the control; by contrast, high concentrations of IAA (11.4 μM) inhibited expression ( Figure 3A). Notably, DlRan3B gene expression showed a sustained decrease as the SA concentration increased ( Figure 3C). In addition, a fluctuating expression pattern was found under MeJA treatment ( Figure 3D). These results indicated that the transcription of DlRan3B responded to plant hormones like auxin, gibberellin, SA, MeJA, and ABA.

The Isolation and Bioinformatic Analysis of the Putative Promoter Region of the DlRan3B Gene
To figure out the regulatory roles of DlRan3B, we further cloned a 1569-bp 5′-flanking sequence upstream of the DlRan3B translation initiation site (ATG), covering a previously cloned 5′-flanking sequence of DlRan3B (714 bp, GenBank: JQ279697). Thereafter, the Berkeley Drosophila Genome Project and PlantCARE databases were used to predict and analyze potential core sequences and regulatory elements of DlRan3B promoter region. Two core sequences in the promoter region were predicted at −952 to −903 (score 0.87) and at −79 to −30 (score 0.97) upstream of the ATG. The potential transcription start sites (TSSs) were T and C, respectively. In the previous study [18], some of the TSSs were proved to be in the region between −79 and −30, but not in the sequence between −952 and −903, thus we proposed that the actual core sequence within the 1569-bp promoter region of DlRan3B was located in the region from −79 to −30 and the actual TSS was C ( Figure 4). Further analysis identified conserved TATA and CAAT boxes dispersed over the entire promoter sequence. The most frequent motifs observed in the DlRan3B promoter were elements involved in light and hormone responses (auxin, gibberellin, SA, MeJA, and ABA). Stress-responsive motifs were

The Effect of Exogenous Plant Hormones on DlRan3B Expression
To understand how DlRan3B responds to plant hormones, we analyzed the relative levels of DlRan3B mRNA in longan embryogenic calluses (ECs) treated with different concentrations of IAA, GA 3 , SA, MeJA, and ABA ( Figure 3A-E). Among the treatments, 26.0 µM GA 3 enhanced the DlRan3B transcript level to approximately 1.6-fold in contrast to the control ( Figure 3B); ABA induced a slight, positive transcriptional control on DlRan3B, with no dose-dependent effects ( Figure 3E). IAA over the range of 2.9-8.6 µM increased DlRan3B expression, with 8.6 µM IAA inducing a 1.3-fold level to the control; by contrast, high concentrations of IAA (11.4 µM) inhibited expression ( Figure 3A). Notably, DlRan3B gene expression showed a sustained decrease as the SA concentration increased ( Figure 3C). In addition, a fluctuating expression pattern was found under MeJA treatment ( Figure 3D). These results indicated that the transcription of DlRan3B responded to plant hormones like auxin, gibberellin, SA, MeJA, and ABA.

The Isolation and Bioinformatic Analysis of the Putative Promoter Region of the DlRan3B Gene
To figure out the regulatory roles of DlRan3B, we further cloned a 1569-bp 5 1 -flanking sequence upstream of the DlRan3B translation initiation site (ATG), covering a previously cloned 5 1 -flanking sequence of DlRan3B (714 bp, GenBank: JQ279697). Thereafter, the Berkeley Drosophila Genome Project and PlantCARE databases were used to predict and analyze potential core sequences and regulatory elements of DlRan3B promoter region. Two core sequences in the promoter region were predicted at´952 to´903 (score 0.87) and at´79 to´30 (score 0.97) upstream of the ATG. The potential transcription start sites (TSSs) were T and C, respectively. In the previous study [18], some of the TSSs were proved to be in the region between´79 and´30, but not in the sequence between´952 and´903, thus we proposed that the actual core sequence within the 1569-bp promoter region of DlRan3B was located in the region from´79 to´30 and the actual TSS was C (Figure 4). Further analysis identified conserved TATA and CAAT boxes dispersed over the entire promoter sequence. The most frequent motifs observed in the DlRan3B promoter were elements involved in light and hormone responses (auxin, gibberellin, SA, MeJA, and ABA). Stress-responsive motifs were also found throughout the promoter region, for example, those involved in low-temperature responsiveness and drought-inducibility ( Figure 4, Table 1). also found throughout the promoter region, for example, those involved in low-temperature responsiveness and drought-inducibility ( Figure 4, Table 1).

Deletion Analysis of the DlRan3B Promoter
To detect the transcription regulation of the DlRan3B promoter and figure out key regulatory regions, the constructs of the 1569-bp flanking fragment and a variety of 5 1 and 3 1 deletions, all fused to GUS, a promoterless reporter gene, were prepared ( Figure 5). Quantitative real-time PCR (qPCR) assays showed that all of the constructs expressed in tobacco leaves produced relatively lower levels of GUS transcripts compared with the control (CaMV35S, 35S) ( Figure 5). The difference of GUS transcripts between each of the two 5 1 deletions showed that activation functional elements were distributed in the positions from´1269 to´924 and´616 to´558, while repression functional elements existed at positions from´1569 to´1269 and´858 to´616. Among the 3 1 deletions, positive elements were distributed in the regions from´1211 to´863 and´245 to´1, while inhibitory ones were at´863 to´245. It indicated that both positive and negative functional elements exist in the DlRan3B promoter region. Table 1. Prediction of regulatory elements in the 5 1 -flanking sequence of DlRan3B.

Regulatory Element
Function Sequence

Responsiveness of the DlRan3B Promoter to Hormone Treatments
The DlRan3B promoter contains certain phytohormone-responsive elements, such as ones involved in auxin (AuxRR-core), gibberellin (GARE-motif), SA (TCA-element), MeJA (TGACG-and CGTCA-motif), and ABA (ABRE) responsiveness. To detect the effects of plant hormones on the transcription regulation of DlRan3B promoter, 8.6 μM IAA, 34.6 μM GA3, 75 μM SA, 100 μM MeJA, and 75.7 μM ABA were used in treating tobacco leaves harboring the construct of DlRan3B promoter. The highest GUS expression was found in samples under IAA treatment, which showed more than 3-fold higher GUS expression compared with the control. GA3 also induced GUS expression, by about 1.5-fold higher than the control. By contrast, GUS expression in the SA-treated samples was repressed. These data demonstrated that regulatory elements related to auxin, gibberellin, and SA responsiveness co-exist in the 1569-bp region of the DlRan3B promoter and they are involved in regulating the transcription of a target gene; meanwhile, MeJA and ABA had no significant effect on the transcription regulation of the DlRan3B promoter ( Figure 6).

Responsiveness of the DlRan3B Promoter to Hormone Treatments
The DlRan3B promoter contains certain phytohormone-responsive elements, such as ones involved in auxin (AuxRR-core), gibberellin (GARE-motif), SA (TCA-element), MeJA (TGACG-and CGTCA-motif), and ABA (ABRE) responsiveness. To detect the effects of plant hormones on the transcription regulation of DlRan3B promoter, 8.6 µM IAA, 34.6 µM GA 3 , 75 µM SA, 100 µM MeJA, and 75.7 µM ABA were used in treating tobacco leaves harboring the construct of DlRan3B promoter. The highest GUS expression was found in samples under IAA treatment, which showed more than 3-fold higher GUS expression compared with the control. GA 3 also induced GUS expression, by about 1.5-fold higher than the control. By contrast, GUS expression in the SA-treated samples was repressed. These data demonstrated that regulatory elements related to auxin, gibberellin, and SA responsiveness co-exist in the 1569-bp region of the DlRan3B promoter and they are involved in regulating the transcription of a target gene; meanwhile, MeJA and ABA had no significant effect on the transcription regulation of the DlRan3B promoter ( Figure 6).

DlRan3B Shares Similar Subcellular Localization with Its Longan Homolog and Animal Counterpart
The result of the subcellular localization assay revealed that DlRan3B was exclusively located in

DlRan3B Shares Similar Subcellular Localization with Its Longan Homolog and Animal Counterpart
The result of the subcellular localization assay revealed that DlRan3B was exclusively located in the nucleus (Figure 1 and Figure S1), slightly different from DlRan3A, another member in the longan Ran family, which was primarily located in the nucleus, with some in the cytoplasm [6]. The nuclear location of DlRan3B is similar with its longan homolog DlRan3A and animal counterpart. Ran is essential for transporting proteins through the nuclear pore in yeast and mammalian tissue [19,20]. DlRan3B might have a similar nucleocytoplasmic trafficking function to its animal counterparts. However, it remains to be verified what is the relationship between DlRan3Bs accumulation and longan late SE, and how DlRan3B functions in embryogenic cells.

DlRan3B Participates in the Auxin, Gibberellin, and ABA Signaling Pathways During Longan Late SE
In the early courses of longan SE, there are high levels of total auxin and gibberellin (IAA + GA 3 ), while longan late SE is associated with lower levels of IAA + GA 3 . There was a slight increase in endogenous ABA as SE progressed in longan; during late SE there was a sharp rise in the ratio of ABA/(IAA + GA 3 ) to a maximum, rather than a distinct increase in the absolute level of ABA, which suggested the critical role of ABA in the late development of longan somatic embryos [21]. Here, we found that lower doses of IAA and GA 3 increased the expression of DlRan3B, while higher doses did not. Therefore, the low levels of DlRan3B transcripts in early longan SE could be attributed to the high levels of auxin and gibberellin. By contrast, DlRan3B expression increased to its maximum at the CE stage, probably because of the complex effects of IAA + GA 3 and ABA. This was different to the expression pattern observed for DlRan3A [6], and suggested that DlRan3B plays a more important role in longan late SE, involving the hormone signaling pathways of auxin, gibberellin, and ABA. However, during longan SE, the exact nature of this complex relationship among auxin, gibberellin, and ABA has not yet been determined.

The Potential Role of DlRan3B
Ran and Ran-binding proteins have important roles in actively transporting various light and phytohormone receptors, transcription factors, and signaling regulatory proteins into the nucleus when plant cells are exposed to light or phytohormones [4]. However, little is uncovered about the roles of phytohormone in the regulatory network of the Ran gene. Similar to DlRan3A [6], in the DlRan3B's 5 1 flanking sequence, there are many elements involved in light responsiveness (ACE, Box 4, Box I, G-box, GAG-motif, GATA-motif, GT1-motif, I-box, Sp1, and TCCC-motif) and elements involved in hormone responsiveness (ABRE, AuxRR-core, CGTCA-motif, GARE-motif, TCA-element, and TGACG-motif). Here, we found that certain concentrations of IAA and GA 3 increased DlRan3B expression and its promoter transcription activity, which suggested that DlRan3B might participate in auxin and gibberellin responsiveness. The auxin-responsive element is located at´1211 to´924 of the DlRan3B promoter region and might be a critical regulatory element for stimulating DlRan3B expression. A novel role of Ran in meristem initiation regulated by auxin signal was observed by overexpressing TaRAN1 in transgenic Arabidopsis and rice, and a close association between Ran and auxin was also discovered in Ran-related proteins [7,22]. Some reports have shown that exogenous GA 3 upregulates the expression of transcription factors for embryogenesis, and induces the regeneration of somatic embryos, with the acceleration of starch hydrolysis by enhancing α-amylase activity [23,24]. Therefore, DlRan3B's response to GA 3 and the existence of an A-box (a sequence conserved in alpha-amylase promoters) in the DlRan3B promoter suggest the probable involvement of gibberellin in activating nucleocytoplasmic transport of signal elements associated with energy metabolism during longan SE. Nevertheless, little direct evidence is available about the relationship between Ran and hormone signaling pathways and about how Ran and its promoter function in plant somatic embryos.
The crucial functions of the nuclear trafficking machinery in plant immune and stress signals were reviewed by García and Parker [5]. SA plays a key role in abiotic stress responses, including drought, low temperature, and salinity responsiveness. Some reports have demonstrated that the Ran protein is involved in abiotic stress responses, such as mechanical wounding [25], osmotic stress [9], salinity [26], and cold stress [10,27]. The observation that SA repressed DlRan3B expression and its promoter transcription activity correlates with the coexistence of elements involved in drought-inducibility and low-temperature responsiveness in the DlRan3B promoter region´858 to´616, which might be negative regulatory elements. Therefore, we hypothesized that SA might participate in the complex interaction of defense-related cis-acting elements and trans-acting factors, and the transport of certain transcription factors or mRNA through the nuclear pore via its effect on the DlRan3B promoter. In addition, the fact that DlRan3B shares ARE (elements involved in anaerobic induction) and TC-rich repeats (elements involved in defense and stress responses) with DlRan3A in its promoter region suggested that the longan Ran gene family might participate in the response to environmental stress by the mutual effects of different family members. Thus, the ARE, TC-rich repeats, MBS (drought-inducibility), LTR (low-temperature responsiveness), TCA-element (SA responsiveness), and some light-responsive elements, which were located in´1569 to´1269,´858 to´616, and´558 tó 245, might be negative regulatory elements. The AuxRR-core (auxin responsiveness), 5 1 UTR Py-rich stretch (high transcription levels conferring), and some other light-responsive elements, which were located at´1211 to´924,´616 to´558, and´245 to´1, might be positive regulatory elements. The MeJA-responsiveness elements might be involved in complex activation and repression regulation because of their scattered pattern in the promoter region. With environmental stimuli, Ran's roles in cell activities and nucleocytoplasmic transport of signal elements in plant embryos require further research.

Subcellular Localization
The subcellular localization assay was conducted as previously described [6]. DlRan3B cDNA was fused to the N-terminus of the GFP in the plasmid of pCAMBIA1302. Transient transformation of fluorescent fusion protein by agro-infiltration in epidermal cells of tobacco (Nicotiana benthamiana) leaves was performed as previously executed and the subcellular localization of DlRan3B-mGFP was analyzed by laser scanning confocal microscopy (Olympus (Tokyo, Japan); FV1200) [6].
Sequences underlined indicate the restriction enzyme sites of HindIII and BamHI.

Phytohormone Treatments of Tobacco Leaves
To examine the effects of phytohormone on the transcription regulation of the DlRan3B promoter, tobacco leaves infiltrated with Agrobacterium harboring DlRan3B promoter were sprayed with 8.6 µM IAA, 34.6 µM GA 3 , 75 µM SA, 100 µM MeJA, or 75.7 µM ABA for 48 h, with sterile water as a control. All tobacco plants were grown and sampled as above described.

qPCR Analysis
qPCR was conducted to investigate the transcript levels of the DlRan3B gene during longan SE, under a series of plant hormone treatments, and transient expression of the GUS gene in agro-infiltrated tobacco leaf samples, as described above. Total RNA extraction, determination, and cDNA synthesis were conducted as described previously [6]. qPCR of the DlRan3B gene was performed using gene-specific primers (F: CATCATGAAGCTGAGCTTGC; R: CCAGCCTGCAACTGTTCTC), with EF-1α, elF-4α, and DlFSD1α as the reference genes [30,34]. Relative transcript levels of the GUS gene were detected using primers (F: CCTGCGTCAATGTAATGTTCTG; R: TTCTCTGCCGTTTCCAAATC), with 18SrRNA as the reference gene of 18SrRNA (F: CCTGAGAAACGGCTACCACAT; R: CACCAGACTTGCCCTCCA).