Comprehensive Analysis of the SBP Family in Blueberry and Their Regulatory Mechanism Controlling Chlorophyll Accumulation

Abstract

SQUAMOSA Promoter Binding Protein (SBP) family genes act as central players to regulate plant growth and development with functional redundancy and specificity. Addressing the diversity of the SBP family in crops is of great significance to precisely utilize them to improve agronomic traits. Blueberry is an important economic berry crop. However, the SBP family has not been described in blueberry. In the present study, twenty VcSBP genes were identified through data mining against blueberry transcriptome databases. These VcSBPs could be clustered into eight groups, and the gene structures and motif compositions are divergent among the groups and similar within each group. The VcSBPs were differentially expressed in various tissues. Intriguingly, 10 VcSBPs were highly expressed at green fruit stages and dramatically decreased at the onset of fruit ripening, implying that they are important regulators during early fruit development. Computational analysis showed that 10 VcSBPs were targeted by miR156, and four of them were further verified by degradome sequencing. Moreover, their functional diversity was studied in Arabidopsis. Noticeably, three VcSBPs significantly increased chlorophyll accumulation, and qRT-PCR analysis indicated that VcSBP13a in Arabidopsis enhanced the expression of chlorophyll biosynthetic genes such as AtDVR, AtPORA, AtPORB, AtPORC, and AtCAO. Finally, the targets of VcSBPs were computationally identified in blueberry, and the Y1H assay showed that VcSBP13a could physically bind to the promoter region of the chlorophyll-associated gene VcLHCB1. Our findings provided an overall framework for individually understanding the characteristics and functions of the SBP family in blueberry.

Molecular characterization of SPL gene family during flower morphogenesis and regulation in blueberry

“…Different plants contain different numbers of VcSPL family members, which seems to be caused by functional redundancy or divergence of SPL family members during evolution. Notably, 24 SPL genes have been previously identified in blueberry [ 53 ], indicating that the number of previously identified blueberry SPL genes was underestimated, and this experiment further complemented the blueberry VcSPL gene family. To investigate the role of VcSBL genes in function, we showed the functional diversity of the blueberry SPL family in terms of phylogeny, gene structure, chromosomal localization, evolutionary history, and spatiotemporal expression.…”

“…To identify SPL-encoding genes in blueberry, we initially performed a local BLAST library build with 16 SPL genes from Arabidopsis thaliana for comparison, followed by a further screening in combination with PFAM scan analysis to remove redundant sequences and then renaming them according to their position on the chromosome, finally identified a total of 56 VcSPL genes (Supplementary Table 1 ). The previous study indicated that 32 blueberry SBP/SPL gene families had been identified and screened, including two alternative SPL alternative splicing [ 53 ]. However, the previous identification of SPL encoding genes was based on a de novo assembly of the transcriptome, which was supplemented by further identification at the level of the whole blueberry genome in this experiment, with 24 new SPL gene families identified, and the relationship between the two SPL identifications corresponds in Supplementary Table 3 .…”

“…However, the previous identification of SPL encoding genes was based on a de novo assembly of the transcriptome, which was supplemented by further identification at the level of the whole blueberry genome in this experiment, with 24 new SPL gene families identified, and the relationship between the two SPL identifications corresponds in Supplementary Table 3 . Two alternative splicings were not identified in the whole genome data, and in the previous study, two pairs of genes ( VcSBP14a and VcSBP14aAS , VcSBP14c , and VcSBP14cAS ) could be derived from different RNA splicing of the same genes [ 53 ]. In this study, VcSBP14aAS and VcSBP14cAS were not identified, while VcSBP14a and VcSBP14c corresponded to VcSPL43 and VcSPL30 , respectively, in the present study.…”

“…Firstly, in evolutionary branching and gene structure, genes belonging to the same subclass had the same motif and structural domain characteristics and were clearly distinguished from other subclasses, for example, motif6 was only present in Group2, presumably, this subclass may play a unique mechanism and function in blueberry. The biological functions of genes were often related to their gene expression patterns and structures [ 53 ]. In the present study, most members contained SPL structural domains, while Ank_2 superfamily structural domains were found in VcSPL27 , VcSPL28 , VcSPL34, and VcSPL43 , and they may be involved in protein interactions [ 55 ].…”

“…Overexpression of OsSPL7 reduced the number of tillers in plants, whereas OsSPL7 RNAi was shown to increase the number of tillers and reduce plant height [ 64 ]. Meanwhile, in the previous study [ 53 ], heterologous expression of VcSPL40 ( VcSBP8b ) was found to exhibit late flowering in Arabidopsis and mi156-targeting VcSPL40 . SPL genes targeted by miR156/157 in Arabidopsis could directly and positively regulate downstream flowering genes, such as SOC1 , LFY , AP1, and FUL [ 18 , 19 ], and GA might also promote flowering by directly binding to SPL transcription factors and activating miR172 and MADS-box genes [ 20 ].…”