SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-box genes encode a family of plant-specific transcription factors (TFs) that play important roles in many growth and development processes including phase transition, leaf initiation, shoot and inflorescence branching, fruit development and ripening etc. The SBP-box gene family has been identified and characterized in many species, but has not been well studied in Petunia, an important ornamental genus. We identified 21 putative SPL genes of Petunia axillaris and P. inflata from the reference genome of P. axillaris N and P. inflata S6, respectively, which were supported by the transcriptome data. For further confirmation, all the 21 genes were also cloned from P. hybrida line W115 (Mitchel diploid). Phylogenetic analysis based on the highly conserved SBP domains arranged PhSPLs in eight groups, analogous to those from Arabidopsis and tomato. Furthermore, the Petunia SPL genes had similar exon-intron structure and the deduced proteins contained very similar conserved motifs within the same subgroup. Out of 21 PhSPL genes, fourteen were predicted to be potential targets of PhmiR156/157, and the putative miR156/157 response elements (MREs) were located in the coding region of group IV, V, VII and VIII genes, but in the 3’-UTR regions of group VI genes. SPL genes were also identified from another two wild Petunia species, P. integrifolia and P. exserta, based on their transcriptome databases to investigate the origin of PhSPLs. Phylogenetic analysis and multiple alignments of the coding sequences of PhSPLs and their orthologs from wild species indicated that PhSPLs were originated mainly from P. axillaris. qRT-PCR analysis demonstrated differential spatiotemperal expression patterns of PhSPL genes in petunia and many were expressed predominantly in the axillary buds and/or inflorescences. In addition, overexpression of PhSPL9a and PhSPL9b in Arabidopsis suggested that these genes play a conserved role in promoting the vegetative-to-reproductive phase transition. Petunia genome contains at least 21 SPL genes, and most of the genes are expressed in different tissues. The PhSPL genes may play conserved and diverse roles in plant growth and development, including flowering regulation, leaf initiation, axillary bud and inflorescence development. This work provides a comprehensive understanding of the SBP-box gene family in Petunia and lays a significant foundation for future studies on the function and evolution of SPL genes in petunia.

MiR156/157 plays multiple pivotal roles during plant growth and development. In this study, we identified 11 miR156- and 5 miR157-encoding loci from the genome of Petunia axillaris and Petunia inflata, designated as PaMIR0156/157s and PiMIR0156/157s, respectively. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis indicated that PhmiR156/157 was expressed predominantly in cotyledons, germinating seeds, flower buds, young fruits and seedlings. PhmiR156/157 levels declined in shoot apical buds and leaves of petunia before flowering as the plant ages; moreover, the temporal expression patterns of most miR156/157-targeted PhSPLs were complementary to that of PhmiR156/157. Ectopic expression of PhMIR0157a in Arabidopsis and petunia resulted in delayed flowering, dwarf plant stature, increased branches and reduced organ size. However, PhMIR0156f-overexpressing Arabidopsis and petunia plants showed only delayed flowering. In addition, downregulation of PhmiR156/157 level by overexpressing STTM156/157 led to taller plants with less branches, longer internodes and precocious flowering. qRT-PCR analysis indicated that PhmiR156/157 modulates these traits mainly by downregulating their PhSPL targets and subsequently decreasing the expression of flowering regulatory genes. Our results demonstrate that the PhmiR156/157-PhSPL module has conserved but also divergent functions in growth and development, which will help us decipher the genetic basis for the improvement of flower transition, plant architecture and organ development in petunia.

The plant-specific TCP transcription factors are well-characterized in both monocots and dicots, which have been implicated in multiple aspects of plant biological processes such as leaf morphogenesis and senescence, lateral branching, flower development and hormone crosstalk. However, no systematic analysis of the petunia TCP gene family has been described. In this work, a total of 66 petunia TCP genes (32 PaTCP genes in P. axillaris and 34 PiTCP genes in P. inflata) were identified. Subsequently, a systematic analysis of 32 PaTCP genes was performed. The phylogenetic analysis combined with structural analysis clearly distinguished the 32 PaTCP proteins into two classes—class Ι and class Ⅱ. Class Ⅱ was further divided into two subclades, namely, the CIN-TCP subclade and the CYC/TB1 subclade. Plenty of cis-acting elements responsible for plant growth and development, phytohormone and/or stress responses were identified in the promoter of PaTCPs. Distinct spatial expression patterns were determined among PaTCP genes, suggesting that these genes may have diverse regulatory roles in plant growth development. Furthermore, differential temporal expression patterns were observed between the large- and small-flowered petunia lines for most PaTCP genes, suggesting that these genes are likely to be related to petal development and/or petal size in petunia. The spatiotemporal expression profiles and promoter analysis of PaTCPs indicated that these genes play important roles in petunia diverse developmental processes that may work via multiple hormone pathways. Moreover, three PaTCP-YFP fusion proteins were detected in nuclei through subcellular localization analysis. This is the first comprehensive analysis of the petunia TCP gene family on a genome-wide scale, which provides the basis for further functional characterization of this gene family in petunia.

Background: The ALOG (Arabidopsis LSH1 and Oryza G1) family of proteins, namely DUF640 (domain of unknown function 640) domain proteins, were found in land plants. Functional characterization of a few ALOG members in model plants such as Arabidopsis and rice suggested they play important regulatory roles in plant development. The information about its evolution, however, is largely limited, and there was no any report on the ALOG genes in Petunia, an important ornamental species. Results: The ALOG genes were identified in four species of Petunia including P. axillaris, P. inflata, P. integrifolia, and P. exserta based on the genome and/or transcriptome databases, which were further confirmed by cloning from P. hybrida ‘W115’ (Mitchel diploid), a popular laboratorial petunia line susceptible to genetic transformation. Phylogenetic analysis indicated that Petunia ALOG genes (named as LSHs according to their closest Arabidopsis homologs) were grouped into four clades, which can be further divided into eight groups, and similar exon-intron structure and motifs are reflected in the same group. The PhLSH genes of hybrid petunia ‘W115’ were mainly derived from P. axillaris. The qPCR analysis revealed distinct spatial expression patterns among them suggesting potentially functional diversification. Moreover, over-expressing PhLSH7a and PhLSH7b in Arabidopsis uncovered their functions in the development of both vegetative and reproductive organs. Conclusions: Petunia genome includes 11 ALOG genes that can be divided into eight distinct groups, and they also show different expression patterns. Among these genes, PhLSH7b and PhLSH7a play significant roles in plant growth and development, especially in fruit development. Our results provide new insight into the evolution of ALOG gene family and have laid a good foundation for the study of petunia LSH gene in the future.