Potato snakin-1 gene silencing affects cell division, primary metabolism, and cell wall composition

NARHIRÑAK, VANESA; ALMASIA, NATALIA INÉS; FERNANDEZ PAULA VIRGINIA; HOPP H. ESTEBAN; ESTEVEZ JOSÉ MANUEL; CARRARI FERNANDO; VAZQUEZ-ROVERE, CECILIA

PLANT PHYSIOLOGY.

AMER SOC PLANT BIOLOGISTS

Año: 2011 vol. 158 p. 252 - 263

0032-0889

Snakin-1 (SN1) is an antimicrobial cysteine-rich peptide isolated from Solanum tuberosum that was classified as a member of the snakin/GASA protein family. In this work, a transgenic approach was used to study the role of SN1 in planta. Even when overexpressing SN1 potato lines did not show remarkable morphological differences from wild type, SN1 silencing resulted in reduced height which was accompanied by an overall reduction in leaf size and severe alterations of leaf shape. Analysis of adaxial epidermis of mature leaves revealed that silenced lines had 70 to 90% increases in the mean cell size with respect to WT leaves. Consequently, the number of epidermal cells was significantly reduced in these lines. Confocal microscopy analysis after agroinfiltration of Nicotiana benthamiana leaves showed that SN1-GFP fusion protein was localized in plasma membrane and bimolecular fluorescence complementation (BIFC) assays revealed that SN1 selfinteracted in vivo. We further focused our study on leaf metabolism by applying a combination of gas chromatography coupled to mass spectrometry (GC-MS), Fourier transform infra red (FT-IR) spectroscopy and spectrophotometric techniques. These targeted analyses allowed a detailed examination of the changes occurring in 46 intermediate compounds from primary metabolic pathways and in 7 cell wall constituents. We demonstrated that SN1 silencing affects cell division, leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. which was accompanied by an overall reduction in leaf size and severe alterations of leaf shape. Analysis of adaxial epidermis of mature leaves revealed that silenced lines had 70 to 90% increases in the mean cell size with respect to WT leaves. Consequently, the number of epidermal cells was significantly reduced in these lines. Confocal microscopy analysis after agroinfiltration of Nicotiana benthamiana leaves showed that SN1-GFP fusion protein was localized in plasma membrane and bimolecular fluorescence complementation (BIFC) assays revealed that SN1 selfinteracted in vivo. We further focused our study on leaf metabolism by applying a combination of gas chromatography coupled to mass spectrometry (GC-MS), Fourier transform infra red (FT-IR) spectroscopy and spectrophotometric techniques. These targeted analyses allowed a detailed examination of the changes occurring in 46 intermediate compounds from primary metabolic pathways and in 7 cell wall constituents. We demonstrated that SN1 silencing affects cell division, leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. remarkable morphological differences from wild type, SN1 silencing resulted in reduced height which was accompanied by an overall reduction in leaf size and severe alterations of leaf shape. Analysis of adaxial epidermis of mature leaves revealed that silenced lines had 70 to 90% increases in the mean cell size with respect to WT leaves. Consequently, the number of epidermal cells was significantly reduced in these lines. Confocal microscopy analysis after agroinfiltration of Nicotiana benthamiana leaves showed that SN1-GFP fusion protein was localized in plasma membrane and bimolecular fluorescence complementation (BIFC) assays revealed that SN1 selfinteracted in vivo. We further focused our study on leaf metabolism by applying a combination of gas chromatography coupled to mass spectrometry (GC-MS), Fourier transform infra red (FT-IR) spectroscopy and spectrophotometric techniques. These targeted analyses allowed a detailed examination of the changes occurring in 46 intermediate compounds from primary metabolic pathways and in 7 cell wall constituents. We demonstrated that SN1 silencing affects cell division, leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. which was accompanied by an overall reduction in leaf size and severe alterations of leaf shape. Analysis of adaxial epidermis of mature leaves revealed that silenced lines had 70 to 90% increases in the mean cell size with respect to WT leaves. Consequently, the number of epidermal cells was significantly reduced in these lines. Confocal microscopy analysis after agroinfiltration of Nicotiana benthamiana leaves showed that SN1-GFP fusion protein was localized in plasma membrane and bimolecular fluorescence complementation (BIFC) assays revealed that SN1 selfinteracted in vivo. We further focused our study on leaf metabolism by applying a combination of gas chromatography coupled to mass spectrometry (GC-MS), Fourier transform infra red (FT-IR) spectroscopy and spectrophotometric techniques. These targeted analyses allowed a detailed examination of the changes occurring in 46 intermediate compounds from primary metabolic pathways and in 7 cell wall constituents. We demonstrated that SN1 silencing affects cell division, leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. classified as a member of the snakin/GASA protein family. In this work, a transgenic approach was used to study the role of SN1 in planta. Even when overexpressing SN1 potato lines did not show remarkable morphological differences from wild type, SN1 silencing resulted in reduced height which was accompanied by an overall reduction in leaf size and severe alterations of leaf shape. Analysis of adaxial epidermis of mature leaves revealed that silenced lines had 70 to 90% increases in the mean cell size with respect to WT leaves. Consequently, the number of epidermal cells was significantly reduced in these lines. Confocal microscopy analysis after agroinfiltration of Nicotiana benthamiana leaves showed that SN1-GFP fusion protein was localized in plasma membrane and bimolecular fluorescence complementation (BIFC) assays revealed that SN1 selfinteracted in vivo. We further focused our study on leaf metabolism by applying a combination of gas chromatography coupled to mass spectrometry (GC-MS), Fourier transform infra red (FT-IR) spectroscopy and spectrophotometric techniques. These targeted analyses allowed a detailed examination of the changes occurring in 46 intermediate compounds from primary metabolic pathways and in 7 cell wall constituents. We demonstrated that SN1 silencing affects cell division, leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. which was accompanied by an overall reduction in leaf size and severe alterations of leaf shape. Analysis of adaxial epidermis of mature leaves revealed that silenced lines had 70 to 90% increases in the mean cell size with respect to WT leaves. Consequently, the number of epidermal cells was significantly reduced in these lines. Confocal microscopy analysis after agroinfiltration of Nicotiana benthamiana leaves showed that SN1-GFP fusion protein was localized in plasma membrane and bimolecular fluorescence complementation (BIFC) assays revealed that SN1 selfinteracted in vivo. We further focused our study on leaf metabolism by applying a combination of gas chromatography coupled to mass spectrometry (GC-MS), Fourier transform infra red (FT-IR) spectroscopy and spectrophotometric techniques. These targeted analyses allowed a detailed examination of the changes occurring in 46 intermediate compounds from primary metabolic pathways and in 7 cell wall constituents. We demonstrated that SN1 silencing affects cell division, leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. remarkable morphological differences from wild type, SN1 silencing resulted in reduced height which was accompanied by an overall reduction in leaf size and severe alterations of leaf shape. Analysis of adaxial epidermis of mature leaves revealed that silenced lines had 70 to 90% increases in the mean cell size with respect to WT leaves. Consequently, the number of epidermal cells was significantly reduced in these lines. Confocal microscopy analysis after agroinfiltration of Nicotiana benthamiana leaves showed that SN1-GFP fusion protein was localized in plasma membrane and bimolecular fluorescence complementation (BIFC) assays revealed that SN1 selfinteracted in vivo. We further focused our study on leaf metabolism by applying a combination of gas chromatography coupled to mass spectrometry (GC-MS), Fourier transform infra red (FT-IR) spectroscopy and spectrophotometric techniques. These targeted analyses allowed a detailed examination of the changes occurring in 46 intermediate compounds from primary metabolic pathways and in 7 cell wall constituents. We demonstrated that SN1 silencing affects cell division, leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. which was accompanied by an overall reduction in leaf size and severe alterations of leaf shape. Analysis of adaxial epidermis of mature leaves revealed that silenced lines had 70 to 90% increases in the mean cell size with respect to WT leaves. Consequently, the number of epidermal cells was significantly reduced in these lines. Confocal microscopy analysis after agroinfiltration of Nicotiana benthamiana leaves showed that SN1-GFP fusion protein was localized in plasma membrane and bimolecular fluorescence complementation (BIFC) assays revealed that SN1 selfinteracted in vivo. We further focused our study on leaf metabolism by applying a combination of gas chromatography coupled to mass spectrometry (GC-MS), Fourier transform infra red (FT-IR) spectroscopy and spectrophotometric techniques. These targeted analyses allowed a detailed examination of the changes occurring in 46 intermediate compounds from primary metabolic pathways and in 7 cell wall constituents. We demonstrated that SN1 silencing affects cell division, leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. Solanum tuberosum that was classified as a member of the snakin/GASA protein family. In this work, a transgenic approach was used to study the role of SN1 in planta. Even when overexpressing SN1 potato lines did not show remarkable morphological differences from wild type, SN1 silencing resulted in reduced height which was accompanied by an overall reduction in leaf size and severe alterations of leaf shape. Analysis of adaxial epidermis of mature leaves revealed that silenced lines had 70 to 90% increases in the mean cell size with respect to WT leaves. Consequently, the number of epidermal cells was significantly reduced in these lines. Confocal microscopy analysis after agroinfiltration of Nicotiana benthamiana leaves showed that SN1-GFP fusion protein was localized in plasma membrane and bimolecular fluorescence complementation (BIFC) assays revealed that SN1 selfinteracted in vivo. We further focused our study on leaf metabolism by applying a combination of gas chromatography coupled to mass spectrometry (GC-MS), Fourier transform infra red (FT-IR) spectroscopy and spectrophotometric techniques. These targeted analyses allowed a detailed examination of the changes occurring in 46 intermediate compounds from primary metabolic pathways and in 7 cell wall constituents. We demonstrated that SN1 silencing affects cell division, leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. which was accompanied by an overall reduction in leaf size and severe alterations of leaf shape. Analysis of adaxial epidermis of mature leaves revealed that silenced lines had 70 to 90% increases in the mean cell size with respect to WT leaves. Consequently, the number of epidermal cells was significantly reduced in these lines. Confocal microscopy analysis after agroinfiltration of Nicotiana benthamiana leaves showed that SN1-GFP fusion protein was localized in plasma membrane and bimolecular fluorescence complementation (BIFC) assays revealed that SN1 selfinteracted in vivo. We further focused our study on leaf metabolism by applying a combination of gas chromatography coupled to mass spectrometry (GC-MS), Fourier transform infra red (FT-IR) spectroscopy and spectrophotometric techniques. These targeted analyses allowed a detailed examination of the changes occurring in 46 intermediate compounds from primary metabolic pathways and in 7 cell wall constituents. We demonstrated that SN1 silencing affects cell division, leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. remarkable morphological differences from wild type, SN1 silencing resulted in reduced height which was accompanied by an overall reduction in leaf size and severe alterations of leaf shape. Analysis of adaxial epidermis of mature leaves revealed that silenced lines had 70 to 90% increases in the mean cell size with respect to WT leaves. Consequently, the number of epidermal cells was significantly reduced in these lines. Confocal microscopy analysis after agroinfiltration of Nicotiana benthamiana leaves showed that SN1-GFP fusion protein was localized in plasma membrane and bimolecular fluorescence complementation (BIFC) assays revealed that SN1 selfinteracted in vivo. We further focused our study on leaf metabolism by applying a combination of gas chromatography coupled to mass spectrometry (GC-MS), Fourier transform infra red (FT-IR) spectroscopy and spectrophotometric techniques. These targeted analyses allowed a detailed examination of the changes occurring in 46 intermediate compounds from primary metabolic pathways and in 7 cell wall constituents. We demonstrated that SN1 silencing affects cell division, leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. which was accompanied by an overall reduction in leaf size and severe alterations of leaf shape. Analysis of adaxial epidermis of mature leaves revealed that silenced lines had 70 to 90% increases in the mean cell size with respect to WT leaves. Consequently, the number of epidermal cells was significantly reduced in these lines. Confocal microscopy analysis after agroinfiltration of Nicotiana benthamiana leaves showed that SN1-GFP fusion protein was localized in plasma membrane and bimolecular fluorescence complementation (BIFC) assays revealed that SN1 selfinteracted in vivo. We further focused our study on leaf metabolism by applying a combination of gas chromatography coupled to mass spectrometry (GC-MS), Fourier transform infra red (FT-IR) spectroscopy and spectrophotometric techniques. These targeted analyses allowed a detailed examination of the changes occurring in 46 intermediate compounds from primary metabolic pathways and in 7 cell wall constituents. We demonstrated that SN1 silencing affects cell division, leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. in planta. Even when overexpressing SN1 potato lines did not show remarkable morphological differences from wild type, SN1 silencing resulted in reduced height which was accompanied by an overall reduction in leaf size and severe alterations of leaf shape. Analysis of adaxial epidermis of mature leaves revealed that silenced lines had 70 to 90% increases in the mean cell size with respect to WT leaves. Consequently, the number of epidermal cells was significantly reduced in these lines. Confocal microscopy analysis after agroinfiltration of Nicotiana benthamiana leaves showed that SN1-GFP fusion protein was localized in plasma membrane and bimolecular fluorescence complementation (BIFC) assays revealed that SN1 selfinteracted in vivo. We further focused our study on leaf metabolism by applying a combination of gas chromatography coupled to mass spectrometry (GC-MS), Fourier transform infra red (FT-IR) spectroscopy and spectrophotometric techniques. These targeted analyses allowed a detailed examination of the changes occurring in 46 intermediate compounds from primary metabolic pathways and in 7 cell wall constituents. We demonstrated that SN1 silencing affects cell division, leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. which was accompanied by an overall reduction in leaf size and severe alterations of leaf shape. Analysis of adaxial epidermis of mature leaves revealed that silenced lines had 70 to 90% increases in the mean cell size with respect to WT leaves. Consequently, the number of epidermal cells was significantly reduced in these lines. Confocal microscopy analysis after agroinfiltration of Nicotiana benthamiana leaves showed that SN1-GFP fusion protein was localized in plasma membrane and bimolecular fluorescence complementation (BIFC) assays revealed that SN1 selfinteracted in vivo. We further focused our study on leaf metabolism by applying a combination of gas chromatography coupled to mass spectrometry (GC-MS), Fourier transform infra red (FT-IR) spectroscopy and spectrophotometric techniques. These targeted analyses allowed a detailed examination of the changes occurring in 46 intermediate compounds from primary metabolic pathways and in 7 cell wall constituents. We demonstrated that SN1 silencing affects cell division, leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. SN1 silencing resulted in reduced height which was accompanied by an overall reduction in leaf size and severe alterations of leaf shape. Analysis of adaxial epidermis of mature leaves revealed that silenced lines had 70 to 90% increases in the mean cell size with respect to WT leaves. Consequently, the number of epidermal cells was significantly reduced in these lines. Confocal microscopy analysis after agroinfiltration of Nicotiana benthamiana leaves showed that SN1-GFP fusion protein was localized in plasma membrane and bimolecular fluorescence complementation (BIFC) assays revealed that SN1 selfinteracted in vivo. We further focused our study on leaf metabolism by applying a combination of gas chromatography coupled to mass spectrometry (GC-MS), Fourier transform infra red (FT-IR) spectroscopy and spectrophotometric techniques. These targeted analyses allowed a detailed examination of the changes occurring in 46 intermediate compounds from primary metabolic pathways and in 7 cell wall constituents. We demonstrated that SN1 silencing affects cell division, leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. SN1 silencing affects cell division, leaf primary metabolism and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. additional roles in growth and development beyond its previously assigned role in plant defense. SN1 has additional roles in growth and development beyond its previously assigned role in plant defense.