TY - BOOK

T1 - Characterization of Phosphate Transporters BdPT4 and BdPT8 in Mycorrhizal and Non-Mychorrhizal Brachypodium distachyon

AU - Clausen, Signe Sandbech

PY - 2016

Y1 - 2016

N2 - Phosphorus (P) is an essential plant nutrient in agricultural production but the P rock reserves used for production of fertilizers are currently being depleted. One approach to reduce the demand on P fertilizers is to optimize the phosphate (Pi) uptake and utilization efficiency of crop plants to support maximal growth at a low nutrient supply.Roots of most plant species are colonized by arbuscular mycorrhiza (AM) fungi, which increase the uptake of nutrients, in particular P. In grasses, however, AM colonization may result in growth depressions, which have conventionally been ascribed to fungal drain of plant carbon. The aim of this thesis was to explore the alternative hypothesis that such growth depressions are caused by Pi limitation under conditions where a repressed direct Pi uptake in AM plants is not fully compensated by the AM-mediated Pi uptake. The hypothesized relationship between P limitation and growth depression was investigated by manipulation of the transcript levels of BdPT4 and BdPT8, two putative direct Brachypodium distachyon phosphate transporters (BdPT). Using a reverse genetic transformation approach, over-expression (OE) lines and knock-down (RNAi) lines of each BdPT gene was generated by Agrobacterium tumefaciens-mediated transformation. The transgenic lines were characterized for effects on growth and plant P uptake, and their subcellular localization was moreover determined by confocal microscopy.Quantitative expression analysis of 11 BdPTs in AM and non-mycorrhizal (NM) control plants confirmed the expression patterns previously observed for nine of the genes in a semi-quantitative study, but differences were found for BdPT2 and BdPT10. The OE lines of both BdPTs were successfully produced, whereas transcript levels in the RNAi lines were significantly repressed only for BdPT4 and not for BdPT8. Colonization of B. distachyon roots by AM fungi resulted in growth depressions in all tested lines including the control line, however OE of either BdPT4 or BdPT8 did not result in improved growth nor P uptake in AM plants. Furthermore, RNAi of BdPT4 had no effect on plant growth nor AM colonization, suggesting that at BdPT4 is redundant. However, the BdPT4 RNAi lines showed a small, but significant reduction in the AM-mediated P uptake and shoot P content, suggesting a role of BdPT4 in AM mediated Pi uptake.The subcellular localization studies confirmed that both BdPT proteins were expressed in cells where direct PTs are believed to perform their function, as well as in the vascular tissue. In NM plants, BdPT4 was localized to the plasma membrane whereas BdPT8 accumulated in the endoplasmic reticulum (ER) in the secretory pathway. A similar expression pattern was observed in non-arbusculated cells of AM plants. In arbusculated cells, BdPT8 was detected in the ER while BdPT4 was localized to the plasma membrane and the periarbuscular membrane (PAM) surrounding the arbuscular trunk and thick branches, which further supported a role of both BdPTs in direct Pi uptake, and for BdPT4 in AM-mediated Pi uptake as well.In conclusion, the studies suggest that BdPT4 and possibly also BdPT8 are involved in direct uptake as well as in translocation of Pi in B. distachyon. Interestingly, as BdPT4 was additionally localised to the PAM of arbusculated cells, BdPT4 may also be involved in uptake of Pi via the AM pathway, which correlates with the slightly decreased AM-mediated uptake in BdPT4-RNAi plants. Since mitigation of AM-related growth depression was not achieved by OE of the assumed direct uptake pathway transporter genes BdPT4 and BdPT8, the alternative hypothesis was not supported in this study, and it was concluded that direct Pi uptake was not limited by the BdPT transcript levels.

AB - Phosphorus (P) is an essential plant nutrient in agricultural production but the P rock reserves used for production of fertilizers are currently being depleted. One approach to reduce the demand on P fertilizers is to optimize the phosphate (Pi) uptake and utilization efficiency of crop plants to support maximal growth at a low nutrient supply.Roots of most plant species are colonized by arbuscular mycorrhiza (AM) fungi, which increase the uptake of nutrients, in particular P. In grasses, however, AM colonization may result in growth depressions, which have conventionally been ascribed to fungal drain of plant carbon. The aim of this thesis was to explore the alternative hypothesis that such growth depressions are caused by Pi limitation under conditions where a repressed direct Pi uptake in AM plants is not fully compensated by the AM-mediated Pi uptake. The hypothesized relationship between P limitation and growth depression was investigated by manipulation of the transcript levels of BdPT4 and BdPT8, two putative direct Brachypodium distachyon phosphate transporters (BdPT). Using a reverse genetic transformation approach, over-expression (OE) lines and knock-down (RNAi) lines of each BdPT gene was generated by Agrobacterium tumefaciens-mediated transformation. The transgenic lines were characterized for effects on growth and plant P uptake, and their subcellular localization was moreover determined by confocal microscopy.Quantitative expression analysis of 11 BdPTs in AM and non-mycorrhizal (NM) control plants confirmed the expression patterns previously observed for nine of the genes in a semi-quantitative study, but differences were found for BdPT2 and BdPT10. The OE lines of both BdPTs were successfully produced, whereas transcript levels in the RNAi lines were significantly repressed only for BdPT4 and not for BdPT8. Colonization of B. distachyon roots by AM fungi resulted in growth depressions in all tested lines including the control line, however OE of either BdPT4 or BdPT8 did not result in improved growth nor P uptake in AM plants. Furthermore, RNAi of BdPT4 had no effect on plant growth nor AM colonization, suggesting that at BdPT4 is redundant. However, the BdPT4 RNAi lines showed a small, but significant reduction in the AM-mediated P uptake and shoot P content, suggesting a role of BdPT4 in AM mediated Pi uptake.The subcellular localization studies confirmed that both BdPT proteins were expressed in cells where direct PTs are believed to perform their function, as well as in the vascular tissue. In NM plants, BdPT4 was localized to the plasma membrane whereas BdPT8 accumulated in the endoplasmic reticulum (ER) in the secretory pathway. A similar expression pattern was observed in non-arbusculated cells of AM plants. In arbusculated cells, BdPT8 was detected in the ER while BdPT4 was localized to the plasma membrane and the periarbuscular membrane (PAM) surrounding the arbuscular trunk and thick branches, which further supported a role of both BdPTs in direct Pi uptake, and for BdPT4 in AM-mediated Pi uptake as well.In conclusion, the studies suggest that BdPT4 and possibly also BdPT8 are involved in direct uptake as well as in translocation of Pi in B. distachyon. Interestingly, as BdPT4 was additionally localised to the PAM of arbusculated cells, BdPT4 may also be involved in uptake of Pi via the AM pathway, which correlates with the slightly decreased AM-mediated uptake in BdPT4-RNAi plants. Since mitigation of AM-related growth depression was not achieved by OE of the assumed direct uptake pathway transporter genes BdPT4 and BdPT8, the alternative hypothesis was not supported in this study, and it was concluded that direct Pi uptake was not limited by the BdPT transcript levels.

UR - https://soeg.kb.dk/permalink/45KBDK_KGL/fbp0ps/alma99122912436105763

M3 - Ph.D. thesis

BT - Characterization of Phosphate Transporters BdPT4 and BdPT8 in Mycorrhizal and Non-Mychorrhizal Brachypodium distachyon

PB - Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen

ER -