SCI1 expression and its transcriptional regulation in the floral meristem and flowers of Nicotiana tabacum

• Autor(a) principal: Cruz, Joelma de Oliveira
• Data de Publicação: 2023
• Tipo de documento: Tese
• Idioma: eng
• Título da fonte: Biblioteca Digital de Teses e Dissertações da USP
• Texto Completo: https://www.teses.usp.br/teses/disponiveis/17/17135/tde-08082023-145223/
• topic: Cell proliferation; Células meristemáticas; Desenvolvimento floral; Floral development; Floral meristem; Meristema floral; Meristematic cells; Proliferação celular; Regulação transcricional; Transcriptional regulation

The reproduction of angiosperms is strictly regulated by genetic pathways and environmental signals that result in the transition from the vegetative to the reproductive phase. The reproductive phase is marked by the floral meristem that results in the flower, a highly modified branch formed by the four floral whorls: sepals, petals, stamens, and pistil. The stamens and the pistil are responsible for producing male and female gametes, therefore, are of great importance in reproduction. Understanding the development of these whorls involves understanding the molecular mechanisms that guarantee correct development. To better understand the development of the pistil, our research group carried out the initial characterization of a gene that was preferentially expressed in the pistil of the Nicotiana tabacum flower and controls proliferation in this organ. This was named SCI1 (Stigma/style Cell-cycle Inhibitor 1). The mechanism of action of SCI1 has not yet been elucidated, and advances in investigations have revealed an extensive network of proteins with which SCI1 interacts. The SCI1 interactome allowed us to assume its involvement in RNA processing and cell cycle, basic processes for cell maintenance. The involvement of SCI1 in these processes allowed us to raise the hypothesis that SCI1 could express itself in other floral whorls and start to express itself in the initial stages of floral development. Therefore, this work aimed to determine where and when the SCI1 gene starts its expression in N. tabacum flowers, relate SCI1 expression to pistil development and analyze the expression of SCI1 in the ovaries analyze the transcriptional regulation of SCI1. To analyze SCI1 expression, in situ hybridization was used. It was observed that SCI1 starts its expression in the floral meristem and remains intensely expressed in the beginnings of floral whorls. Expression of SCI1 in floral meristem and whorl primordia indicates its involvement in the development of all floral whorls. As the whorls are specified, the expression of SCI1 is reduced, except in the pistil, the organ in which the last meristematic cells of the flower are located. SCI1 mRNA was detected both in non-fused and already fused carpels. SCI1 is also detected in the young ovary when the placenta is expanding. It is later detected in the primordia of ovules and ovules with their structures, integument funiculus, and nucellus established. In these structures, SCI1 was detected in the funiculus and integument. In situ hybridization also revealed co-expression of SCI1 with the NAG1, NtANT, and NtWUS genes. SCI1 co-expressed with NtWUS and NAG1 in the floral meristem. NtWUS encodes a Homeobox transcription factor that regulates the proliferation of pluripotent cells in the floral meristem, and NAG1 encodes a transcription factor responsible for the specification of the third and fourth floral whorls and the termination of meristematic cells. In stamens and carpels, SCI1 is co-expressed with NAG1 and NtANT. SCI1 co-expresses with NtANT in ovule primordia, integument, and funiculus. NtANT encodes a transcription factor with the AP2 domain that positively regulates cell proliferation from the beginning of floral development and acts on the correct development of the pistil and ovules. The co-expression of SCI1 with the transcription factors NtWUS, NAG1, and NtANT, added to the function described for SCI1, which involves controlling cell proliferation and controlling stigma and style development, reveals a possible regulation of SCI1 by these transcription factors. These transcription factors interactions with cis-elements in the SCI1 promoter were predicted in silico and confirmed in monohybrid assays (Yeast One Hybrid) with the portion of the SCI1 promoter, called Frag1, which comprises 443pb above the initiation codon (ATG). Interactions were also confirmed via EMSA. With luciferase assay, it was possible to determine what effect the interaction of these transcription factors has on SCI1 expression. Transcription factors NAG1 and NtANT promote activation of SCI1 expression by binding to their respective cis-elements. While the interaction of NtWUS was demonstrated to reduce the expression of SCI1, how results were not expressive. The involvement of auxin in the regulation of SCI1 was also predicted. It was demonstrated by the luciferase assay that the synthetic phytohormone NAA significantly increases the expression of SCI1 when regulated by NtANT. The IIA phytohormone positively influenced SCI1 expression when regulated by NtANT. The endogenous expression of the SCI1 protein was obtained through transgenic plants expressing the SCI1 protein in translational fusion to GFP under the control of its endogenous promoter. Thus, it was possible to determine the protein\'s location in the flower. Like mRNA, the SCI1 protein is detected from the floral meristem and in all young whorls. A centripetal protein reduction was observed as the whorls developed, but this reduction was not observed in the pistil until stage 2. Due to the size of the flower, observations at more advanced stages were impossible. It was also possible to observe the SCI1 protein in specialized tissues of the stigma and style, transmitting tissue of the style and secretory zone of the stigma and in the parenchyma. In the ovaries, the SCI1 protein was detected in the placenta and the ovules in their integument and funiculus. With these plants, it was possible to confirm the location of SCI1 in the nucleus and nucleolus. The data set confirms the hypothesis that SCI1 begins to express itself in the floral meristem. Furthermore, the data also point to the expression of SCI1 in meristematic cells. Regulation of SCI1 by NtWUS reinforces its relationship with meristematic cells in the floral meristem. While the presence of SCI1 in the pistil and its regulation by the transcription factors NAG1 and NtANT reinforce the involvement of SCI1 in the proliferation of meristematic cells still present in these organs.