PtoRFL30,a Mitochondrion-localized Protein,regulates Secondary Vascular Development Of Stem In Poplar

Wood formation depends on the secondary xylem development of stems in trees,which is driven by the activity of vascular cambium.The differentiated daughter cells of cambium undergo cell expansion,secondary wall deposition,programmed cell death,and finally form the mature woody tissues.The program of wood formation has been known to be precisely regulated by the complex networks involving phytohormones and transcription factors.The process of wood formation also requires the production of energy.However,the molecular mechanism of how energy signals effect the secondary development of trees remains unclear.Mitochondria are the site of oxidative metabolism of eukaryotic cells,where sugars,are finally catabolized to release energy.Recent studies have revealed that mitochondrial signaling plays important roles in plant development and responses to environmental stimuli.As a type of semi-autonomous organelle,mitochondria contain their own genetic materials,but the expression of mitochondrion-encoding genes requires the help of a large number of mitochondrial-localized proteins encoded by nuclear genes.These proteins are usually involved in the post-transcriptional processing in mitochondria.Pentatricopeptide repeat（PPR）proteins are one of the key regulators of RNA metabolism in mitochondria.PPR proteins usually consist of 2-27 PPR motives that are arranged in tandem.A series of mitochondrial-localized PPR proteins have been demonstrated to play important roles in the post-transcriptional processes of mitochondrion-encoding genes,including RNA processing,intron splicing,editing,and translation.PPR proteins regulate mitochondrial homeostasis through modulating mitochondrial gene expression,and thereby affect the seed fertility,stress response and other developmental processes in Arabidopsis,maize,rice,and Petunia etc.However,the function of PPR proteins in woody plants,especially during secondary development,still needs to be further explored.In order to explore the function of PPR proteins in secondary development of woody plants,we identified a P-type PPR protein（named PtoRFL30）through bioinformatics analysis,and found that the number of cambium and xylem cell layers was strongly compromised in the PtoRFL30-overexpressing transgenic plants.These results indicated that PtoRFL30 is involved in the secondary vascular development.Hence,the poplar was used to elucidate the molecular mechanism of PtoRFL30 regulating secondary vascular development in stems and the essential downstream signaling pathway.The main results are as follow:1.To explore the regulatory role of PPR proteins in secondary vascular development via mitochondrial function,a P-type PPR protein which may be localized in mitochondria and highly expressed in stem was selected by bioinformatics analysis and RT-q PCR,and named as PtoRFL30 based on its chromosomal location.2.To validate the tissue-specific expression patterns of PtoRFL30,RT-q PCR and GUS activity analyses were performed.PtoRFL30 was more strongly expressed in stems and young leaves than in roots,mature leaves and petioles.And the assays of GUS activity driven by its promoter indicated that the PtoRFL30 is preferentially expressed in the secondary vascular tissues,especially in the cambial zone,and weakly in root tips and leaf vein.These results suggested that PtoRFL30 regulates the secondary vascular development in poplar.3.To identify the role of PtoRFL30 in regulating wood formation,we constructed the overexpressing and RNA interfering（RNAi）vectors of PtoRFL30,and obtained the transgenic poplar plants.Phenotypic observation showed that the cambium and xylem of PtoRFL30-overexpressing lines had less cell layers than WT plants at the same internodes,while the PtoRFL30-RNAi plants exhibited the opposite tendency.These results indicated that PtoRFL30 may play a negative role in the secondary development of poplar.4.In other species,the RFL genes encode mitochondrion-localized PPR proteins which suppress the expression of mitochondrion-encoding genes.To further investigate the role of PtoRFL30 in mitochondrial functions,the expression of mitochondrion-encoding genes in PtoRFL30 transgenic plants were examined by semi-quantitative PCR and RT-q PCR analysis.The results showed that the relative expression levels of most mitochondrial-encoded genes in PtoRFL30-overexpressing and RNAi lines exhibited significant differences compared with that in WT plants.The biochemical parametes of mitochondrial functions were examined.The PtoRFL30-overexpressing plants had the lowest H₂O₂ and ATP content,followed by the wild-type plants,and the RNAi plants had the highest content of H₂O₂ and ATP.As validated by DAB and NBT staining,these results indicated that overexpression of PtoRFL30 disrupts the mitochondrial functions.5.To investigate whether mitochondrial modulation is required for the PtoRFL30-mediated secondary development in poplar,the mitochondrial inhibitors,including rotenone and antimycin A,were used to treat the WT and PtoRFL30-RNAi plants.The results showed that secondary development of both WT and PtoRFL30-RNAi plants was suppressed after rotenone and antimycin A treatment,indicating that mitochondrial function is required for the PtoRFL30-mediated secondary vascular development.6.To reveal the downstream signal of mitochondrion-mediated secondary development,RNAseq analyese were used to investigate the comparative transcriptome between PtoRFL30-overexpressing and WT plants.Compared with the WT plants,differentially expressed genes（DEGs）were identified including hormone-related genes,in which the auxin-related genes account for a large proportion.The results of RT-q PCR were in consistent with RNA-seq,and revealed that the auxin pathway participate in the PtoRFL30-directed mitochondrial regulation of secondary development in poplar.7.To further verify the hypothesis,the auxin content of PtoRFL30 transgenic plants were determined by LC-MS.The overexpression of PtoRFL30 caused the reduction of auxin content.Hence,the auxin homeostasis was compromised by treating PtoRFL30-RNAi and PtoRFL30-overexpression plants with 1-N-naphthylphthalamic acid（NPA）and 1-Naphthylacetic acid（NAA）,respectively.The number of cambial and xylem cell layers were increased after NAA treatment in PtoRFL30-overexpressing plants compared with the control,while the number of cambial and xylem cell layers were significantly reduced after NPA treatment in the PtoRFL30-RNAi plants.At the same time,the transgenic plants with impaired mitochondrial functions were treated with NAA.By analyzing the phenotypes of secondary vascular development,we found that NAA could restore the phenotype of secondary developmental defects caused by the destruction of mitochondrial functions.These results suggested that auxin is involved in the regulation of secondary development by mitochondria.In summary,these results showed that PtoRFL30-mediated secondary vascular development requires mitochondrial regulation,and this pathway depends on auxin.This study provided novel insights into the understanding of wood formation in trees and mitochondrial regulation on plant development.