Genome-wide identification, evolution, and role of SPL gene family in beet (Beta vulgaris L.) under cold stress.

BACKGROUND: SPL transcription factors play vital roles in regulating plant growth, development, and abiotic stress responses. Sugar beet (Beta vulgaris L.), one of the world's main sugar-producing crops, is a major source of edible and industrial sugars for humans. Although the SPL gene family has been extensively identified in other species, no reports on the SPL gene family in sugar beet are available. RESULTS: Eight BvSPL genes were identified at the whole-genome level and were renamed based on their positions on the chromosome. The gene structure, SBP domain sequences, and phylogenetic relationship with Arabidopsis were analyzed for the sugar beet SPL gene family. The eight BvSPL genes were divided into six groups (II, IV, V, VI, VII, and VIII). Of the BvSPL genes, no tandem duplication events were found, but one pair of segmental duplications was present. Multiple cis-regulatory elements related to growth and development were identified in the 2000-bp region upstream of the BvSPL gene start codon (ATG). Using quantitative real-time polymerase chain reaction (qRT-PCR), the expression profiles of the eight BvSPL genes were examined under eight types of abiotic stress and during the maturation stage. BvSPL transcription factors played a vital role in abiotic stress, with BvSPL3 and BvSPL6 being particularly noteworthy. CONCLUSION: Eight sugar beet SPL genes were identified at the whole-genome level. Phylogenetic trees, gene structures, gene duplication events, and expression profiles were investigated. The qRT-PCR analysis indicated that BvSPLs play a substantial role in the growth and development of sugar beet, potentially participating in the regulation of root expansion and sugar accumulation.

Genome-wide identification and expression analysis of the SPL transcription factor family and its response to abiotic stress in Quinoa (Chenopodium quinoa).

BACKGROUND: Squamous promoter binding protein-like (SPL) proteins are a class of transcription factors that play essential roles in plant growth and development, signal transduction, and responses to biotic and abiotic stresses. The rapid development of whole genome sequencing has enabled the identification and characterization of SPL gene families in many plant species, but to date this has not been performed in quinoa (Chenopodium quinoa). RESULTS: This study identified 23 SPL genes in quinoa, which were unevenly distributed on 18 quinoa chromosomes. Quinoa SPL genes were then classified into eight subfamilies based on homology to Arabidopsis thaliana SPL genes. We selected three dicotyledonous and monocotyledonous representative species, each associated with C. quinoa, for comparative sympatric mapping to better understand the evolution of the developmental mechanisms of the CqSPL family. Furthermore, we also used 15 representative genes from eight subfamilies to characterize CqSPLs gene expression in different tissues and at different fruit developmental stages under six different abiotic stress conditions. CONCLUSIONS: This study, the first to identify and characterize SPL genes in quinoa, reported that CqSPL genes, especially CqSPL1, play a critical role in quinoa development and in its response to various abiotic stresses.

Genome-wide identification, phylogeny and expression analysis of the SPL gene family and its important role in salt stress in Medicago sativa L.

BACKGROUND: SQUAMOSA promoter-binding protein-like (SPL) transcription factors are widely present in plants and are involved in signal transduction, the stress response and development. The SPL gene family has been characterized in several model species, such as A. thaliana and G. max. However, there is no in-depth analysis of the SPL gene family in forage, especially alfalfa (Medicago sativa L.), one of the most important forage crops worldwide. RESULT: In total, 76 putative MsSPL genes were identified in the alfalfa genome with an uneven distribution. Based on their identity and gene structure, these MsSPLs were divided into eight phylogenetic groups. Seventy-three MsSPL gene pairs arose from segmental duplication events, and the MsSPLs on the four subgenomes of individual chromosomes displayed high collinearity with the corresponding M. truncatula genome. The prediction of the cis-elements in the promoter regions of the MsSPLs detected two copies of ABA (abscisic acid)-responsive elements (ABREs) on average, implying their potential involvement in alfalfa adaptation to adverse environments. The transcriptome sequencing of MsSPLs in roots and leaves revealed that 54 MsSPLs were expressed in both tissues. Upon salt treatment, three MsSPLs (MsSPL17, MsSPL23 and MsSPL36) were significantly regulated, and the transcription level of MsSPL36 in leaves was repressed to 46.6% of the control level. CONCLUSION: In this study, based on sequence homology, we identified 76 SPL genes in the alfalfa. The SPLs with high identity shared similar gene structures and motifs. In total, 71.1% (54 of 76) of the MsSPLs were expressed in both roots and leaves, and the majority (74.1%) preferred underground tissues to aerial tissues. MsSPL36 in leaves was significantly repressed under salt stress. These findings provide comprehensive information regarding the SPB-box gene family for improve alfalfa tolerance to high salinity.

Cone-setting in spruce is regulated by conserved elements of the age-dependent flowering pathway.

Reproductive phase change is well characterized in angiosperm model species, but less studied in gymnosperms. We utilize the early cone-setting acrocona mutant to study reproductive phase change in the conifer Picea abies (Norway spruce), a gymnosperm. The acrocona mutant frequently initiates cone-like structures, called transition shoots, in positions where wild-type P. abies always produces vegetative shoots. We collect acrocona and wild-type samples, and RNA-sequence their messenger RNA (mRNA) and microRNA (miRNA) fractions. We establish gene expression patterns and then use allele-specific transcript assembly to identify mutations in acrocona. We genotype a segregating population of inbred acrocona trees. A member of the SQUAMOSA BINDING PROTEIN-LIKE (SPL) gene family, PaSPL1, is active in reproductive meristems, whereas two putative negative regulators of PaSPL1, miRNA156 and the conifer specific miRNA529, are upregulated in vegetative and transition shoot meristems. We identify a mutation in a putative miRNA156/529 binding site of the acrocona PaSPL1 allele and show that the mutation renders the acrocona allele tolerant to these miRNAs. We show co-segregation between the early cone-setting phenotype and trees homozygous for the acrocona mutation. In conclusion, we demonstrate evolutionary conservation of the age-dependent flowering pathway and involvement of this pathway in regulating reproductive phase change in the conifer P. abies.

Genome-wide identification, gene cloning, subcellular location and expression analysis of SPL gene family in P. granatum L.

BACKGROUNDS: Pomegranate is an excellent tree species with nutritional, medicinal, ornamental and ecological values. Studies have confirmed that SPL factors play an important role in floral transition and flower development. RESULTS: Used bioinformatics methods, 15 SPL (SQUAMOSA promoter-binding protein-like) genes were identified and analyzed from the 'Taishanhong' pomegranate (P. granatum L.) genome. Phylogenetic analysis showed that PgSPLs were divided into six subfamilies (G1 ~ G6). PgSPL promoter sequences contained multiple cis-acting elements associated with abiotic stress or hormonal response. Based on the transcriptome data, expression profiles of different tissues and different developmental stages showed that PgSPL genes had distinct temporal and spatial expression characteristics. The expression analysis of miR156 in small RNA sequencing results showed that miR156 negatively regulated the expression of target genes. qRT-PCR analysis showed that the expression levels of PgSPL2, PgSPL3, PgSPL6, PgSPL11 and PgSPL14 in leaves were significantly higher than those in buds and stems (p < 0.05). The expression levels of PgSPL5, PgSPL12 and PgSPL13 in flower buds were significantly higher than that in leaves and stems (p < 0.05). The full-length of coding sequence of PgSPL5 and PgSPL13 were obtained by homologous cloning technology. The full length of PgSPL5 is 1020 bp, and PgSPL13 is 489 bp, which encodes 339 and 162 amino acids, respectively. Further investigation revealed that PgSPL5 and PgSPL13 proteins were located in the nucleus. Exogenous plant growth regulator induction experiments showed that PgSPL5 was up-regulated in leaves and stems. PgSPL13 was up-regulated in leaves and down-regulated in stems. When sprayed with 6-BA, IBA and PP333 respectively, PgSPL5 and PgSPL13 were up-regulated most significantly at P2 (bud vertical diameter was 5.1 ~ 12.0 mm) stage of bisexual and functional male flowers. CONCLUSIONS: Our findings suggested that PgSPL2, PgSPL3, PgSPL6, PgSPL11 and PgSPL14 played roles in leaves development of pomegranate. PgSPL5, PgSPL12 and PgSPL13 played roles in pomegranate flower development. PgSPL5 and PgSPL13 were involved in the response process of different plant hormone signal transduction in pomegranate development. This study provided a robust basis for further functional analyses of SPL genes in pomegranate.

Roles of transcription factor SQUAMOSA promoter binding protein-like gene family in papaya (Carica papaya) development and ripening.

SQUAMOSA promoter binding protein-like (SPL) family plays vital regulatory roles in plant growth and development. The SPL family in climacteric fruit Carica papaya has not been reported. This study identified 14 papaya SPLs (CpSPL) from papaya genome and analyzed their sequence features, phylogeny, intron/exon structure, conserved motif, miR156-mediated posttranscriptional regulation, and expression patterns. 14 CpSPLs were clustered into 8 groups, and two distinct expression patterns were revealed for miR156-targeted and nontargeted CpSPLs in different tissues and fruit development stages. The expression changes of CpSPLs in ethephon and 1-MCP treated fruit during ripening suggested that the CpSPLs guided by CpmiR156 play crucial roles in ethylene signaling pathway. This study sheds light on the new function of SPL family in fruit development and ripening, providing insights on understanding evolutionary divergence of the members of SPL family among plant species.

Genome-wide identification, phylogeny and expression analysis of the SPL gene family in wheat.

BACKGROUND: Members of the plant-specific SPL gene family (squamosa promoter-binding protein -like) contain the SBP conserved domain and are involved in the regulation of plant growth and development, including the development of plant flowers and plant epidermal hair, the plant stress response, and the synthesis of secondary metabolites. This family has been identified in various plants. However, there is no systematic analysis of the SPL gene family at the genome-wide level of wheat. RESULTS: In this study, 56 putative TaSPL genes were identified using the comparative genomics method; we renamed them TaSPL001 - TaSPL056 on their chromosomal distribution. According to the un-rooted neighbor joining phylogenetic tree, gene structure and motif analyses, the 56 TaSPL genes were divided into 8 subgroups. A total of 81 TaSPL gene pairs were designated as arising from duplication events and 64 interacting protein branches were identified as involve in the protein interaction network. The expression patterns of 21 randomly selected TaSPL genes in different tissues (roots, stems, leaves and inflorescence) and under 4 treatments (abscisic acid, gibberellin, drought and salt) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). CONCLUSIONS: The wheat genome contains 56 TaSPL genes and those in same subfamily share similar gene structure and motifs. TaSPL gene expansion occurred through segmental duplication events. Combining the results of transcriptional and qRT-PCR analyses, most of these TaSPL genes were found to regulate inflorescence and spike development. Additionally, we found that 13 TaSPLs were upregulated by abscisic acid, indicating that TaSPL genes play a positive role in the abscisic acid-mediated pathway of the seedling stage. This study provides comprehensive information on the SPL gene family of wheat and lays a solid foundation for elucidating the biological functions of TaSPLs and improvement of wheat yield.

Molecular characterization and expression analysis of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE gene family in Paeonia suffruticosa.

KEY MESSAGE: A total of 16 PsSPL genes were identified in tree peony. PsSPLs potentially regulated flowering time, lateral bud and seed development, and the juvenile-to-adult phase transition. SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors are important for plant growth and development. Here, we report the identification of 16 full-length PsSPLs in tree peony (Peaonia suffruticosa Andr.) and 9 PsSPLs that have miR156 target sites. Phylogenetic analysis of the relationship of SPLs in P. suffruticosa and Arabidopsis suggested that they can be classified into six groups, and PsSPLs were highly correlated with Arabidopsis SPLs counterparts in the same group. Cis-element of promoter region analysis suggested that PsSPL genes play roles in physiological processes and developmental events. Expression analysis indicated that most PsSPL genes exhibited high expression levels in the tissues and organs examined here. The increasing expression levels of PsSPL1, PsSPL2, PsSPL8, PsSPL9, PsSPL12, and PsSPL16, and decreasing expression levels of PsSPL1A and PsSPL1B in buds over time suggested that they were probably regulated by the juvenile-to-adult phase transition. In addition, the expression profiles of PsSPL genes in different developmental buds and seeds suggested that PsSPL2, PsSPL3, PsSPL9, PsSPL10, PsSPL13, and PsSPL13A were important genes for regulating the flowering time of the tree peony; PsSPL2 and PsSPL8 might play a role in suppressing lateral bud development, and PsSPL2, PsSPL13, and PsSPL14 positively controlled grain size and number, and pod branching. These results provide a foundation for future functional analysis of PsSPL genes in tree peony growth and development.

Roles of the SPL gene family and miR156 in the salt stress responses of tamarisk (Tamarix chinensis).

BACKGROUND: Accumulating evidences show that SPLs are crucial regulators of plant abiotic stress tolerance and the highly conserved module miR156/SPL appears to balance plant growth and stress responses. The halophyte Tamarix chinensis is highly resistant to salt tress. SPLs of T. chinensis (TcSPLs) and theirs roles in salt stress responses remain elusive. RESULTS: In this study, we conducted a systematic analysis of the TcSPLs gene family including 12 members belonging to 7 groups. The physicochemical properties and conserved motifs showed divergence among groups and similarity in each group. The microRNA response elements (MREs) are conserved in location and sequence, with the exception of first MRE within TcSPL5. The miR156-targeted SPLs are identified by dual-luciferase reporter assay of MRE-miR156 interaction. The digital expression gene profiles cluster suggested potential different functions of miR156-targeted SPLs vs non-targeted SPLs in response to salt stress. The expression patterns analysis of miR156-targeted SPLs with a reverse expression trend to TcmiR156 suggested 1 h (salt stress time) could be a critical time point of post-transcription regulation in salt stress responses. CONCLUSIONS: Our work demonstrated the post-transcription regulation of miR156-targeted TcSPLs and transcription regulation of non-targeted TcSPLs in salt stress responses, and would be helpful to expound the miR156/SPL-mediated molecular mechanisms underlying T. chinensis salt stress tolerance.

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.

[Identification of CjSPL gene family in Ziyang Xiangcheng rootstock and expression pattern analysis].

Squamosa promoter binding protein-like (SPL) family is a group of important transcription factors involved in the regulation of plant growth and development and the response to environmental stress, but there are few studies in perennial fruit trees such as citrus. In this study, Ziyang Xiangcheng (Citrus junos Sib.ex Tanaka), an important rootstock of Citrus, was used as the material for analysis. Based on plantTFDB transcription factor database and sweet orange genome database, 15 SPL family members were genome-widely identified and cloned from Ziyang Xiangcheng, and named CjSPL1-CjSPL15. Sequence analysis showed that the open reading frame (ORF) length of CjSPLs ranged from 393 bp to 2 865 bp, encoding 130-954 amino acids. Phylogenetic tree divided 15 CjSPLs into 9 subfamilies. Gene structure and conserved domain analysis predicted 20 different conserved motifs and SBP basic domains. Analysis of cis-acting promoter elements predicted 20 different promoter elements, including those related to plant growth and development, abiotic stress and secondary metabolites. The expression patterns of CjSPLs under drought, salt and low temperature stresses were analyzed by real-time fluorescence quantitative PCR (qRT-PCR), and many CjSPLs were significantly up-regulated after stress treatment. This study provides a reference for further study on the function of SPL family transcription factors in citrus and other fruit trees.

The NtSPL Gene Family in Nicotiana tabacum: Genome-Wide Investigation and Expression Analysis in Response to Cadmium Stress.

The SQUAMOSA promoter binding protein-like (SPL)SPL family genes play an important role in regulating plant growth and development, synthesis of secondary metabolites, and resistance to stress. Understanding of the role of the SPL family in tobacco is still limited. In this study, 42 NtSPL genes were identified from the genome of the tobacco variety TN90. According to the results of the conserved motif and phylogenetic tree, the NtSPL genes were divided into eight subgroups, and the genes in the same subgroup showed similar gene structures and conserved domains. The cis-acting element analysis of the NtSPL promoters showed that the NtSPL genes were regulated by plant hormones and stresses. Twenty-eight of the 42 NtSPL genes can be targeted by miR156. Transcriptome data and qPCR results indicated that the expression pattern of miR156-targeted NtSPL genes was usually tissue specific. The expression level of miR156 in tobacco was induced by Cd stress, and the expression pattern of NtSPL4a showed a significant negative correlation with that of miR156. These results suggest that miR156-NtSPL4a may mediate the tobacco response to Cd stress. This study lays a foundation for further research on the function of the NtSPL gene and provides new insights into the involvement of NtSPL genes in the plant response to heavy metal stress.

Comprehensive analyses of the SPL transcription factor family in Paulownia fortunei and their responses to biotic and abiotic stresses.

To study the molecular characteristics, phylogenetic evolution, and gene functions of the SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) genes in Paulownia fortunei, a whole genome sequence analysis was carried out, and a total of 23 PfSPL genes were identified. Tandem duplication and fragment replication were the main patterns of gene expansion in the PfSPL family. Phylogenetic analysis showed that the 23 identified PfSPLs formed seven subgroups, and the structures of the proteins in the same subgroup were similar. Functional analysis indicated that PfSPL11 may regulate flowering, PfSPL5 was involved in gibberellin signaling, PfSPL1/4/23 regulated branching, and PfSPL9/16/18 were related to pathogen resistance. Yeast one hybrid technology confirmed that PfSPL4 and PfSP23 can bind to the promoter of PfTCPa. The transcriptome analysis indicated that PfSPL10 was sensitive to both drought and salt stress. Ten PfSPLs that responded to phytoplasma infection were identified. Molecular docking showed that PfSPL10 and PfSPL 4/5/9/10/11/13 formed active pockets in the conserved SBP domain that could bind methyl methane sulfonate (MMS) and rifampicin (Rif) through stable hydrogen bonds, respectively. This study provides a basis for further studies on the functions of the PfSPL transcription factor family, and for genetic improvement and breeding of trees resistant to PaWB disease.

Genome-Wide Identification, Characterization, and Expression Profiling Analysis of SPL Gene Family during the Inflorescence Development in Trifolium repens.

Trifolium repens is the most widely cultivated perennial legume forage in temperate region around the world. It has rich nutritional value and good palatability, seasonal complementarity with grasses, and can improve the feed intake and digestibility of livestock. However, flowering time and inflorescence development directly affects the quality and yield of T. repens, as well as seed production. The Squa promoter binding protein-like (SPL) gene family is a plant specific transcription factor family, which has been proved to play a critical role in regulating plant formation time and development of flowers. In this study, a total of 37 TrSPL genes were identified from the whole genome of T. repens and were divided into nine clades based on phylogenetic tree. Seventeen TrSPL genes have potential target sites for miR156. The conserved motif of squamosa promoter binding protein (SBP) contains two zinc finger structures and one NLS structure. Gene structure analysis showed that all TrSPL genes contained SBP domain, while ankyrin repeat region was just distributed in part of genes. 37 TrSPL genes were relatively dispersedly distributed on 16 chromosomes, and 5 pairs of segmental repeat genes were found, which indicated that segmental duplication was the main way of gene expansion. Furthermore, the gene expression profiling showed that TrSPL11, TrSPL13, TrSPL22, and TrSPL26 were highly expressed only in the early stage of inflorescence development, while TrSPL1 and TrSPL6 are highly expressed only in the mature inflorescence. Significantly, the expression of TrSPL4 and TrSPL12 increased gradually with the development of inflorescences. The results of this study will provide valuable clues for candidate gene selection and elucidating the molecular mechanism of T. repens flowering regulation.

Genome-Wide Identification and Characterization of SPL Family Genes in Chenopodium quinoa.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes encode a large family of plant-specific transcription factors that play important roles in plant growth, development, and stress responses. However, there is little information available on SPL genes in Chenopodiaceae. Here, 23 SPL genes were identified and characterized in the highly nutritious crop Chenopodium quinoa. Chromosome localization analysis indicated that the 23 CqSPL genes were unevenly distributed on 12 of 18 chromosomes. Two zinc finger-like structures and a nuclear location signal were present in the SBP domains of all CqSPLs, with the exception of CqSPL21/22. Phylogenetic analysis revealed that these genes were classified into eight groups (group I-VIII). The exon-intron structure and motif composition of the genes in each group were similar. Of the 23 CqSPLs, 13 were potential targets of miR156/7. In addition, 5 putative miR156-encoding loci and 13 putative miR157-encoding loci were predicted in the quinoa genome, and they were unevenly distributed on chromosome 1-4. The expression of several Cqu-MIR156/7 loci was confirmed by reverse transcription polymerase chain reaction in seedlings. Many putative cis-elements associated with light, stress, and phytohormone responses were identified in the promoter regions of CqSPLs, suggesting that CqSPL genes are likely involved in the regulation of key developmental processes and stress responses. Expression analysis revealed highly diverse expression patterns of CqSPLs among tissues. Many CqSPLs were highly expressed in leaves, flowers, and seeds, and their expression levels were low in the roots, suggesting that CqSPLs play distinct roles in the development and growth of quinoa. The expression of 13 of 23 CqSPL genes responded to salt treatment (11 up-regulated and 2 down-regulated). A total of 22 of 23 CqSPL genes responded to drought stress (21 up-regulated and 1 down-regulated). Moreover, the expression of 14 CqSPL genes was significantly altered following cadmium treatment (3 up-regulated and 11 down-regulated). CqSPL genes are thus involved in quinoa responses to salt/drought and cadmium stresses. These findings provide new insights that will aid future studies of the biological functions of CqSPLs in C. quinoa.

Comparative analysis of the SPL gene family in five Rosaceae species: Fragaria vesca, Malus domestica, Prunus persica, Rubus occidentalis, and Pyrus pyrifolia.

SQUAMOSA promoter-binding protein-like (SPL) transcription factors are very important for the plant growth and development. Here 15 RoSPLs were identified in Rubus occidentalis. The conserved domains and motifs, phylogenetic relationships, posttranscriptional regulation, and physiological function of the 92 SPL family genes in Fragaria vesca, Malus domestica, Prunus persica, R. occidentalis, and Pyrus pyrifolia were analyzed. Sequence alignment and phylogenetic analysis showed the SPL proteins had sequence conservation, some FvSPLs could be lost or developed, and there was a closer relationship between M. domestica and P. pyrifolia, F. vesca and R. occidentalis, respectively. Genes with similar motifs clustering together in the same group had their functional redundancy. Based on the function of SPLs in Arabidopsis thaliana, these SPLs could be involved in vegetative transition from juvenile to adult, morphological change in the reproductive phase, anthocyanin biosynthesis, and defense stress. Forty-eight SPLs had complementary sequences of miR156, of which nine PrpSPLs in P. persica and eight RoSPLs in R. occidentalis as the potential targets of miR156 were reported for the first time, suggesting the conservative regulatory effects of miR156 and indicating the roles of miR156-SPL modules in plant growth, development, and defense response. It provides a basic understanding of SPLs in Rosaceae plants.

Molecular Characterization of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) Gene Family in Betula luminifera.

As a major family of plant-specific transcription factors, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes play vital regulatory roles in plant growth, development and stress responses. In this study, 18 SPL genes were identified and cloned from Betula luminifera. Two zinc finger-like structures and a nuclear location signal (NLS) segments were existed in the SBP domains of all BlSPLs. Phylogenetic analysis showed that these genes were clustered into nine groups (group I-IX). The intron/exon structure and motif composition were highly conserved within the same group. 12 of the 18 BlSPLs were experimentally verified as the targets of miR156, and two cleavage sites were detected in these miR156-targeted BlSPL genes. Many putative cis-elements, associated with light, stresses and phytohormones response, were identified in the promoter regions of BlSPLs, suggesting that BlSPL genes are probably involved in important physiological processes and developmental events. Tissue-specific expression analysis showed that miR156-targeted BlSPLs exhibited a more differential expression pattern, while most miR156-nontargeted BlSPLs tended to be constitutively expressed, suggesting the distinct roles of miR156-targeted and nontargeted BlSPLs in development and growth of B. luminifera. Further expression analysis revealed that miR156-targeted BlSPLs were dramatically up-regulated with age, whereas mature BlmiR156 level was apparently declined with age, indicating that miR156/SPL module plays important roles in vegetative phase change of B. luminifera. Moreover, yeast two-hybrid assay indicated that several miR156-targeted and nontargeted BlSPLs could interact with two DELLA proteins (BlRGA and BlRGL), which suggests that certain BlSPLs take part in the GA regulated processes through protein interaction with DELLA proteins. All these results provide an important basis for further exploring the biological functions of BlSPLs in B. luminifera.