Genome-Wide Characterization of SPL Gene Family in Codonopsis pilosula Reveals the Functions of CpSPL2 and CpSPL10 in Promoting the Accumulation of Secondary Metabolites and Growth of C. pilosula Hairy Root

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors play critical roles in regulating diverse aspects of plant growth and development, including vegetative phase change, plant architecture, anthocyanin accumulation, lateral root growth, etc. In the present study, 15 SPL genes were identified based on the genome data of Codonopsis pilosula, a well-known medicinal plant. Phylogenetic analysis clustered CpSPLs into eight groups (G1-G8) along with SPLs from Arabidopsis thaliana, Solanum lycopersicum, Oryza sativa and Physcomitrella patens. CpSPLs in the same group share similar gene structure and conserved motif composition. Cis-acting elements responding to light, stress and phytohormone widely exist in their promoter regions. Our qRT-PCR results indicated that 15 CpSPLs were differentially expressed in different tissues (root, stem, leaf, flower and calyx), different developmental periods (1, 2 and 3 months after germination) and various conditions (NaCl, MeJA and ABA treatment). Compared with the control, overexpression of CpSPL2 or CpSPL10 significantly promoted not only the growth of hairy roots, but also the accumulation of total saponins and lobetyolin. Our results established a foundation for further investigation of CpSPLs and provided novel insights into their biological functions. As far as we know, this is the first experimental research on gene function in C. pilosula.

Comparative analysis of the SPL genes among four Ipomoea species and the evidence for a role of some IbSPLs in storage root development in sweet potato (Ipomoea batatas)

Background As a major family of plant-specific transcription factors, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes play crucial regulatory roles in plant growth, development, and stress tolerance. SPL transcription factor family has been widely studied in various plant species, however, there are no systematic studies on SPL genes in genus Ipomoea. Results In this study, a total of 29, 27, 26, 23 SPL genes were identified in Ipomoea batatas, Ipomoea trifida, Ipomoea triloba, and Ipomoea nil, respectively. Phylogenetic analysis indicated that Ipomoea SPL genes could be clustered into eight clades. SPL members within the same clade showed similar gene structures, domain organizations, and cis-acting element compositions, suggesting similarity of biological function potentially. Evolutionary analysis revealed that segmental duplication events played a major role in the Ipomoea genus-specific expansion of SPL genes. Of these Ipomoea SPL genes, 69 were predicted as the target genes of miR156, and 7 IbSPL genes were further confirmed by degradome data. Additionally, IbSPL genes showed diverse expression patterns in various tissues, implying their functional conservation and divergence. Finally, by combining the information from expression patterns and regulatory sub-networks, we found that four IbSPL genes (IbSPL16/IbSPL17/IbSPL21/IbSPL28) may be involved in the formation and development of storage roots. Conclusions This study not only provides novel insights into the evolutionary and functional divergence of the SPL genes in all available sequenced species in genus Ipomoea, but also lays a foundation for further elucidation of the potential functional roles of IbSPL genes during storage root development.

Cloning, Characterization, and Overexpression in Arabidopsis to Determine the Function of SPL Genes from Woodland Strawberry (Fragaria Vesca)

SQUAMOSA promoter binding protein-like (SPL) proteins is a class of plant specific transcription factors that play important roles during plant development. However, the majority of SPL genes in strawberry are functionally uncharacterized. In this study, three SPL genes, i.e. FvSPL1, FvSPL2, and FvSPL11 (FvSPL1/2/11), from woodland strawberry were cloned and characterized. Phylogenetic analysis with SPL genes from Arabidopsis, tomato and chrysanthemum indicated that FvSPL1/2/11 were clustered into the same group with those of miR156 target site located at the 3’-untranslated region (UTR). Further biochemical analysis indicated that FvSPL1 was exclusively localized in the nucleus. Electrophoretic mobility shift assay demonstrated FvSPL1 could specifically recognized the GTAC motif. Transcriptional activity analysis showed FvSPL1 was a transcriptional activator that could activate the expression of FvAP1 gene. Finally, all of the transgenic Arabidopsis that overexpression the three FvSPL genes were exhibited significantly early flowering phenotype. Taken together, our study indicated that FvSPL1/2/11 similar to their orthologs in Arabidopsis mainly functions in regulating plant flowering. These results enriched our understanding to the functions of SPL genes in strawberry and might be utilized for strawberry flowering time manipulation in the future.

Golden orb-weaving spider (Trichonephila clavipes) silk genes with sex-biased expression and atypical architectures

Spider silks are renowned for their high-performance mechanical properties. Contributing to these properties are proteins encoded by the spidroin (spider fibroin) gene family. Spidroins have been discovered mostly through cDNA studies of females based on the presence of conserved terminal regions and a repetitive central region. Recently, genome sequencing of the golden orb-web weaver, Trichonephila clavipes, provided a complete picture of spidroin diversity. Here, we refine the annotation of T. clavipes spidroin genes including the reclassification of some as non-spidroins. We rename these non-spidroins as spidroin-like (SpL) genes because they have repetitive sequences and amino acid compositions like spidroins, but entirely lack the archetypal terminal domains of spidroins. Insight into the function of these spidroin and SpL genes was then examined through tissue- and sex-specific gene expression studies. Using qPCR, we show that some silk genes are upregulated in male silk glands compared to females, despite males producing less silk in general. We also find that an enigmatic spidroin that lacks a spidroin C-terminal domain is highly expressed in silk glands, suggesting that spidroins could assemble into fibers without a canonical terminal region. Further, we show that two SpL genes are expressed in silk glands, with one gene highly evolutionarily conserved across species, providing evidence that particular SpL genes are important to silk production. Together, these findings challenge long-standing paradigms regarding the evolutionary and functional significance of the proteins and conserved motifs essential for producing spider silks.

Arabidopsis NUCLEAR FACTOR Y A8 inhibits the juvenile-to-adult transition by activating transcription of MIR156s

Vegetative (juvenile-to-adult) and flowering (vegetative-to-reproductive) phase changes are crucial in the life cycle of higher plants. MicroRNA156 (miR156) and its target SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes are master regulators that determine vegetative phase changes. The miR156 level gradually declines as a plant ages and its expression is rapidly repressed by sugar. However, the underlying regulatory mechanism of transcriptional regulation of the MIR156 gene remains largely unknown. In this study, we demonstrated that Arabidopsis NUCLEAR FACTOR Y A8 (NF-YA8) binds directly to CCAAT cis-elements in the promoters of multiple MIR156 genes, thus activating their transcription and inhibiting the juvenile-to-adult transition. NF-YA8 was highly expressed in juvenile-stage leaves, and significantly repressed with developmental age and by sugar signals. Our results suggest that NF-YA8 acts as a signaling hub, integrating internal developmental age and sugar signals to regulate the transcription of MIR156s, thus affecting the juvenile-to-adult and flowering transitions.

A Regulatory Network for miR156-SPL Module in Arabidopsis thaliana

Vegetative phase changes in plants describes the transition between juvenile and adult phases of vegetative growth before flowering. It is one of the most fundamental mechanisms for plants to sense developmental signals, presenting a complex process involving many still-unknown determinants. Several studies in annual and perennial plants have identified the conservative roles of miR156 and its targets, SBP/SPL genes, in guiding the switch of plant growth from juvenile to adult phases. Here, we review recent progress in understanding the regulation of miR156 expression and how miR156-SPLs mediated plant age affect other processes in Arabidopsis. Powerful high-throughput sequencing techniques have provided rich data to systematically study the regulatory mechanisms of miR156 regulation network. From this data, we draw an expanded miR156-regulated network that links plant developmental transition and other fundamental biological processes, gaining novel and broad insight into the molecular mechanisms of plant-age-related processes in Arabidopsis.

miR156-targeted SPL10 controls Arabidopsis root meristem activity and root-derived de novo shoot regeneration via cytokinin responses

Root growth is modulated by different factors, including phytohormones, transcription factors, and microRNAs (miRNAs). MicroRNA156 and its targets, the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, define an age-dependent pathway that controls several developmental processes, including lateral root emergence. However, it remains unclear whether miR156-regulated SPLs control root meristem activity and root-derived de novo shoot regeneration. Here, we show that MIR156 and SPL genes have opposing expression patterns during the progression of primary root (PR) growth in Arabidopsis, suggesting that age cues may modulate root development. Plants with high miR156 levels display reduced meristem size, resulting in shorter primary root (PRs). Conversely, plants with reduced miR156 levels show higher meristem activity. Importantly, loss of function of SPL10 decreases meristem activity, while SPL10 de-repression increases it. Meristem activity is regulated by SPL10 probably through the reduction of cytokinin responses, via the modulation of type-B ARABIDOPSIS RESPONSE REGULATOR1(ARR1) expression. We also show that SPL10 de-repression in the PRs abolishes de novo shoot regenerative capacity by attenuating cytokinin responses. Our results reveal a cooperative regulation of root meristem activity and root-derived de novo shoot regeneration by integrating age cues with cytokinin responses via miR156-targeted SPL10.

Molecular Cloning and Characterization of SQUAMOSA-Promoter Binding Protein-Like Gene FvSPL10 from Woodland Strawberry (Fragaria vesca)

SQUAMOSA-promoter binding protein-like (SPL) proteins are plant-specific transcript factors that play essential roles in plant growth and development. Although many SPL genes have been well characterized in model plants like Arabidopsis, rice and tomato, the functions of SPLs in strawberry are still largely elusive. In the present study, we cloned and characterized FvSPL10, the ortholog of AtSPL9, from woodland strawberry. Subcellular localization shows FvSPL10 localizes in the cell nucleus. The luciferase system assay indicates FvSPL10 is a transcriptional activator, and both in vitro and in vivo assays indicate FvSPL10 could bind to the promoter of FvAP1 and activate its expression. Ectopic expression of FvSPL10 in Arabidopsis promotes early flowering and increases organs size. These results demonstrate the multiple regulatory roles of FvSPL10 in plant growth and development and lay a foundation for investigating the biological functions of FvSPL10 in strawberry.

Expression Pattern of FT/TFL1 and miR156-Targeted SPL Genes Associated with Developmental Stages in Dendrobium catenatum

Time to flower, a process either referring to juvenile–adult phase change or vegetative–reproductive transition, is strictly controlled by an intricate regulatory network involving at least both FT/TFL1 and the micro RNA (miR)156-regulated SPL family members. Despite substantial progresses recently achieved in Arabidopsis and other plant species, information regarding the involvement of these genes during orchid development and flowering competence is still limited. Dendrobium catenatum, a popular orchid species, exhibits a juvenile phase of at least three years. Here, through whole-genome mining and whole-family expression profiling, we analyzed the homologous genes of FT/TFL1, miR156, and SPL with special reference to the developmental stages. The FT/TFL1 family contains nine members; among them, DcHd3b transcribes abundantly in young and juvenile tissues but not in adult, contrasting with the low levels of others. We also found that mature miR156, encoded by a single locus, accumulated in large quantity in protocorms and declined by seedling development, coincident with an increase in transcripts of three of its targeted SPL members, namely DcSPL14, DcSPL7, and DcSPL18. Moreover, among the seven predicted miR156-targeted SPLs, only DcSPL3 was significantly expressed in adult plants and was associated with plant maturation. Our results might suggest that the juvenile phase change or maturation in this orchid plant likely involves both the repressive action of a TFL1-like pathway and the promotive effect from an SPL3-mediated mechanism.

Roles of the GA-mediated SPL Gene Family and miR156 in the Floral Development of Chinese Chestnut (Castanea mollissima)

Chestnut (Castanea mollissima) is a deciduous tree species with major economic and ecological value that is widely used in the study of floral development in woody plants due its monoecious and out-of-proportion characteristics. Squamosa promoter-binding protein-like (SPL) is a plant-specific transcription factor that plays an important role in floral development. In this study, a total of 18 SPL genes were identified in the chestnut genome, of which 10 SPL genes have complementary regions of CmmiR156. An analysis of the phylogenetic tree of the squamosa promoter-binding protein (SBP) domains of the SPL genes of Arabidopsis thaliana, Populus trichocarpa, and C. mollissima divided these SPL genes into eight groups. The evolutionary relationship between poplar and chestnut in the same group was similar. A structural analysis of the protein-coding regions (CDSs) showed that the domains have the main function of SBP domains and that other domains also play an important role in determining gene function. The expression patterns of CmmiR156 and CmSPLs in different floral organs of chestnut were analyzed by real-time quantitative PCR. Some CmSPLs with similar structural patterns showed similar expression patterns, indicating that the gene structures determine the synergy of the gene functions. The application of gibberellin (GA) and its inhibitor (Paclobutrazol, PP333) to chestnut trees revealed that these exert a significant effect on the number and length of the male and female chestnut flowers. GA treatment significantly increased CmmiR156 expression and thus significantly decreased the expression of its target gene, CmSPL6/CmSPL9/CmSPL16, during floral bud development. This finding indicates that GA might indirectly affect the expression of some of the SPL target genes through miR156. In addition, RNA ligase-mediated rapid amplification of the 5′ cDNA ends (RLM-RACE) experiments revealed that CmmiR156 cleaves CmSPL9 and CmSPL16 at the 10th and 12th bases of the complementary region. These results laid an important foundation for further study of the biological function of CmSPLs in the floral development of C. mollissima.