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Molecular characterization of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene family from Citrus and the effect of fruit load on their expression.

Shalom L, Shlizerman L, Zur N, Doron-Faigenboim A, Blumwald E, Sadka A - Front Plant Sci (2015)

Bottom Line: We showed that the Citrus SPL was able promote flowering independently of photoperiod in Arabidopsis, while miR156 repressed its flowering-promoting activity.Results showed that two additional SPL-like genes and miR172, known to be induced by SPLs in Arabidopsis, were altered by fruit load.The relationships between these factors in relation to the fruit-load effect on Citrus flowering are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Fruit Tree Sciences, Agricultural Research Organization, The Volcani Center Bet Dagan, Israel ; The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem Rehovot, Israel.

ABSTRACT
We recently identified a Citrus gene encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor that contained a sequence complementary to miR156. Genes of the SPL family are known to play a role in flowering regulation and phase transition. In Citrus, the mRNA levels of the gene were significantly altered by fruit load in buds; under heavy fruit load (ON-Crop trees), known to suppress next year flowering, the mRNA levels were down-regulated, while fruit removal (de-fruiting), inducing next-year flowering, resulted in its up-regulation. In the current work, we set on to study the function of the gene. We showed that the Citrus SPL was able promote flowering independently of photoperiod in Arabidopsis, while miR156 repressed its flowering-promoting activity. In order to find out if fruit load affected the expression of additional genes of the SPL family, we identified and classified all SPL members in the Citrus genome, and studied their seasonal expression patterns in buds and leaves, and in response to de-fruiting. Results showed that two additional SPL-like genes and miR172, known to be induced by SPLs in Arabidopsis, were altered by fruit load. The relationships between these factors in relation to the fruit-load effect on Citrus flowering are discussed.

No MeSH data available.


Genomic structure ofCiSPL5(Ciclev10009879) and its long antisense transcript. Vertical gray lines and triangle tips indicate transcription start site or polyadenylation site identified by 5′ or 3′ RACE. Exon–intron organization of Ciclev10009133 (PP2C) primary and alternative transcripts is based on the Citrus clementina genome database (http://www.phytozome.net/).
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Figure 3: Genomic structure ofCiSPL5(Ciclev10009879) and its long antisense transcript. Vertical gray lines and triangle tips indicate transcription start site or polyadenylation site identified by 5′ or 3′ RACE. Exon–intron organization of Ciclev10009133 (PP2C) primary and alternative transcripts is based on the Citrus clementina genome database (http://www.phytozome.net/).

Mentions: The roles of SPLs and miR156 as regulators of phase transitions have been extensively studied in Arabidopsis and other annual plant species; however, considerably less research has been done with trees. To gain insight into the roles of SPLs in Citrus and understand whether the presence of a miR156-binding site in an SPL transcript constitutes a real regulatory element, we performed a molecular and functional characterization of one Citrus SPL, Ciclev10009879, whose expression had been previously studied in relation to fruit-load effect on flowering induction of Citrus (Shalom et al., 2012, 2014). Full-length sequencing of Ciclev10009879 mRNA revealed that it is 843 bp long (with one alternative polyadenylation site at 750 bp) with a putative ORF encoding a 130-amino acid protein and a miR156-binding site located in the 3′UTR. An antisense transcript (AST) of about 2400 bp which encompasses the entire region of Ciclev10009879 was also identified (Figure 3). Surprisingly, RACE analyses indicated that the transcription start site of this AST is located in a neighboring upstream gene, Ciclev10009133, encoding a putative PROTEIN PHOSPHATASE 2C (PP2C). In fact, the full-length structure of the AST was similar to one of the predicted alternative transcripts of Ciclev10009133, only with a longer than predicted 3′ tail (long PP2C transcript, Figure 3). The RACE analyses identified four transcription start sites and four polyadenylation sites in the AST, suggesting a complex mode of transcription. In Citrus, fruit set takes place during May, whereas September-October are regarded as the last time points at which fruit removal during the ON-year reverses the AB trend (Martinez-Fuentes et al., 2010). The floral induction period starts in mid-November and lasts until approximately mid-January (Davenport, 1990). Expression analysis of the long AST in buds showed that it was expressed at higher levels from May to September and lower levels from November to January (Supplementary Figure 1). However, no significant differences were detected between buds of ON- and OFF-Crop trees, and no alterations were detected following fruit removal, putting its role in flowering control by fruit load into question. As mentioned above, Ciclev10009133 encoded a protein that is highly homologous to Arabidopsis PP2C (At3g15260; 79% identity). In the Arabidopsis genome, this gene is located jointly and in antisense orientation to AtSPL5, which belongs to the small SPLs subgroup (Figure 2). Therefore, based on SBP-domain sequence homology, protein length, miR156-binding site position and genomic coupling with PP2C, we determined that Citrus SPL Ciclev10009879 is the ortholog of Arabidopsis AtSPL5, and it is henceforth referred to as CiSPL5.


Molecular characterization of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene family from Citrus and the effect of fruit load on their expression.

Shalom L, Shlizerman L, Zur N, Doron-Faigenboim A, Blumwald E, Sadka A - Front Plant Sci (2015)

Genomic structure ofCiSPL5(Ciclev10009879) and its long antisense transcript. Vertical gray lines and triangle tips indicate transcription start site or polyadenylation site identified by 5′ or 3′ RACE. Exon–intron organization of Ciclev10009133 (PP2C) primary and alternative transcripts is based on the Citrus clementina genome database (http://www.phytozome.net/).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4443640&req=5

Figure 3: Genomic structure ofCiSPL5(Ciclev10009879) and its long antisense transcript. Vertical gray lines and triangle tips indicate transcription start site or polyadenylation site identified by 5′ or 3′ RACE. Exon–intron organization of Ciclev10009133 (PP2C) primary and alternative transcripts is based on the Citrus clementina genome database (http://www.phytozome.net/).
Mentions: The roles of SPLs and miR156 as regulators of phase transitions have been extensively studied in Arabidopsis and other annual plant species; however, considerably less research has been done with trees. To gain insight into the roles of SPLs in Citrus and understand whether the presence of a miR156-binding site in an SPL transcript constitutes a real regulatory element, we performed a molecular and functional characterization of one Citrus SPL, Ciclev10009879, whose expression had been previously studied in relation to fruit-load effect on flowering induction of Citrus (Shalom et al., 2012, 2014). Full-length sequencing of Ciclev10009879 mRNA revealed that it is 843 bp long (with one alternative polyadenylation site at 750 bp) with a putative ORF encoding a 130-amino acid protein and a miR156-binding site located in the 3′UTR. An antisense transcript (AST) of about 2400 bp which encompasses the entire region of Ciclev10009879 was also identified (Figure 3). Surprisingly, RACE analyses indicated that the transcription start site of this AST is located in a neighboring upstream gene, Ciclev10009133, encoding a putative PROTEIN PHOSPHATASE 2C (PP2C). In fact, the full-length structure of the AST was similar to one of the predicted alternative transcripts of Ciclev10009133, only with a longer than predicted 3′ tail (long PP2C transcript, Figure 3). The RACE analyses identified four transcription start sites and four polyadenylation sites in the AST, suggesting a complex mode of transcription. In Citrus, fruit set takes place during May, whereas September-October are regarded as the last time points at which fruit removal during the ON-year reverses the AB trend (Martinez-Fuentes et al., 2010). The floral induction period starts in mid-November and lasts until approximately mid-January (Davenport, 1990). Expression analysis of the long AST in buds showed that it was expressed at higher levels from May to September and lower levels from November to January (Supplementary Figure 1). However, no significant differences were detected between buds of ON- and OFF-Crop trees, and no alterations were detected following fruit removal, putting its role in flowering control by fruit load into question. As mentioned above, Ciclev10009133 encoded a protein that is highly homologous to Arabidopsis PP2C (At3g15260; 79% identity). In the Arabidopsis genome, this gene is located jointly and in antisense orientation to AtSPL5, which belongs to the small SPLs subgroup (Figure 2). Therefore, based on SBP-domain sequence homology, protein length, miR156-binding site position and genomic coupling with PP2C, we determined that Citrus SPL Ciclev10009879 is the ortholog of Arabidopsis AtSPL5, and it is henceforth referred to as CiSPL5.

Bottom Line: We showed that the Citrus SPL was able promote flowering independently of photoperiod in Arabidopsis, while miR156 repressed its flowering-promoting activity.Results showed that two additional SPL-like genes and miR172, known to be induced by SPLs in Arabidopsis, were altered by fruit load.The relationships between these factors in relation to the fruit-load effect on Citrus flowering are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Fruit Tree Sciences, Agricultural Research Organization, The Volcani Center Bet Dagan, Israel ; The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem Rehovot, Israel.

ABSTRACT
We recently identified a Citrus gene encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor that contained a sequence complementary to miR156. Genes of the SPL family are known to play a role in flowering regulation and phase transition. In Citrus, the mRNA levels of the gene were significantly altered by fruit load in buds; under heavy fruit load (ON-Crop trees), known to suppress next year flowering, the mRNA levels were down-regulated, while fruit removal (de-fruiting), inducing next-year flowering, resulted in its up-regulation. In the current work, we set on to study the function of the gene. We showed that the Citrus SPL was able promote flowering independently of photoperiod in Arabidopsis, while miR156 repressed its flowering-promoting activity. In order to find out if fruit load affected the expression of additional genes of the SPL family, we identified and classified all SPL members in the Citrus genome, and studied their seasonal expression patterns in buds and leaves, and in response to de-fruiting. Results showed that two additional SPL-like genes and miR172, known to be induced by SPLs in Arabidopsis, were altered by fruit load. The relationships between these factors in relation to the fruit-load effect on Citrus flowering are discussed.

No MeSH data available.