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RNAi-based functional elucidation of PtrPRP, a gene encoding a hybrid proline rich protein, in cold tolerance of Poncirus trifoliata.

Peng T, Jia MM, Liu JH - Front Plant Sci (2015)

Bottom Line: PtrPRP is constitutively expressed in root, stem and leaf, with the highest expression levels in leaf.When challenged by low temperature, the PtrPRP-RNAi plants displayed more sensitive performance compared with wild type (WT), as shown by higher electrolyte leakage and malondialdehyde content.In addition, the RNAi lines accumulated more reactive oxygen species (ROS) and lower levels of proline relative to WT.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Horticultural Plant Biology, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan China ; National Navel Orange Engineering Research Center, College of Navel Orange, Gannan Normal University, Ganzhou China.

ABSTRACT
Hybrid proline-rich proteins (HyPRPs) have been suggested to play important roles in various plant development and stress response. In this study, we report the cloning and functional analysis of PtrPRP, a HyPRP-encoding gene of Poncirus trifoliata. PtrPRP contains 176 amino acids, among which 21% are proline residues, and has an 8-cysteine motif (8 CM) domain at the C terminal, a signal peptide and a proline-rich region at the N terminal. PtrPRP is constitutively expressed in root, stem and leaf, with the highest expression levels in leaf. It was progressively induced by cold, but transiently upregulated by salt and ABA. Transgenic P. trifoliata plants with knock-down PtrPRP by RNA interference (RNAi) were generated to investigate the role of PtrPRP in cold tolerance. When challenged by low temperature, the PtrPRP-RNAi plants displayed more sensitive performance compared with wild type (WT), as shown by higher electrolyte leakage and malondialdehyde content. In addition, the RNAi lines accumulated more reactive oxygen species (ROS) and lower levels of proline relative to WT. These results suggested that PtrPRP might be positively involved in cold tolerance by maintaining membrane integrity and ROS homeostasis.

No MeSH data available.


Related in: MedlinePlus

Analysis of proline levels in WT and two RNAi lines after cold treatment. Asterisks indicate that the values of corresponding transgenic lines are statistically significantly different from that of WT (*P < 0.05).
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Figure 9: Analysis of proline levels in WT and two RNAi lines after cold treatment. Asterisks indicate that the values of corresponding transgenic lines are statistically significantly different from that of WT (*P < 0.05).

Mentions: Electrolyte leakage and MDL are indirect indices for oxidative stress that is primarily caused by excessive accumulation of ROS. The drastic difference in EL and MDA levels between RNAi lines and WT prompted us to check ROS status of the tested lines after cold treatment. We first used histochemical staining with DAB and NBT to reveal in situ accumulation of H2O2 and O2-, respectively, in the cold treated leaves. This method is valid as the ROS levels can be directly disclosed based on the color of reaction. As shown in Figure 8A, conspicuous difference in the staining patterns was observed between WT and the RNAi lines. The leaves of RNAi lines were stained by both DAB and NBT in deeper manner or the areas of staining were larger, indicating that the RNAi lines produced more ROS after cold treatment compared with the WT. The staining was partly confirmed by quantitative measurement of H2O2 using a specific kit designed for it (Figure 8B). We also measured proline contents in the RNAi lines and WT after cold treatment, as this compound has been considered as an important metabolite indicating the relevance to stress tolerance. As shown in Figure 9, the WT accumulated more proline in comparison with the two RNAi lines.


RNAi-based functional elucidation of PtrPRP, a gene encoding a hybrid proline rich protein, in cold tolerance of Poncirus trifoliata.

Peng T, Jia MM, Liu JH - Front Plant Sci (2015)

Analysis of proline levels in WT and two RNAi lines after cold treatment. Asterisks indicate that the values of corresponding transgenic lines are statistically significantly different from that of WT (*P < 0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4587090&req=5

Figure 9: Analysis of proline levels in WT and two RNAi lines after cold treatment. Asterisks indicate that the values of corresponding transgenic lines are statistically significantly different from that of WT (*P < 0.05).
Mentions: Electrolyte leakage and MDL are indirect indices for oxidative stress that is primarily caused by excessive accumulation of ROS. The drastic difference in EL and MDA levels between RNAi lines and WT prompted us to check ROS status of the tested lines after cold treatment. We first used histochemical staining with DAB and NBT to reveal in situ accumulation of H2O2 and O2-, respectively, in the cold treated leaves. This method is valid as the ROS levels can be directly disclosed based on the color of reaction. As shown in Figure 8A, conspicuous difference in the staining patterns was observed between WT and the RNAi lines. The leaves of RNAi lines were stained by both DAB and NBT in deeper manner or the areas of staining were larger, indicating that the RNAi lines produced more ROS after cold treatment compared with the WT. The staining was partly confirmed by quantitative measurement of H2O2 using a specific kit designed for it (Figure 8B). We also measured proline contents in the RNAi lines and WT after cold treatment, as this compound has been considered as an important metabolite indicating the relevance to stress tolerance. As shown in Figure 9, the WT accumulated more proline in comparison with the two RNAi lines.

Bottom Line: PtrPRP is constitutively expressed in root, stem and leaf, with the highest expression levels in leaf.When challenged by low temperature, the PtrPRP-RNAi plants displayed more sensitive performance compared with wild type (WT), as shown by higher electrolyte leakage and malondialdehyde content.In addition, the RNAi lines accumulated more reactive oxygen species (ROS) and lower levels of proline relative to WT.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Horticultural Plant Biology, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan China ; National Navel Orange Engineering Research Center, College of Navel Orange, Gannan Normal University, Ganzhou China.

ABSTRACT
Hybrid proline-rich proteins (HyPRPs) have been suggested to play important roles in various plant development and stress response. In this study, we report the cloning and functional analysis of PtrPRP, a HyPRP-encoding gene of Poncirus trifoliata. PtrPRP contains 176 amino acids, among which 21% are proline residues, and has an 8-cysteine motif (8 CM) domain at the C terminal, a signal peptide and a proline-rich region at the N terminal. PtrPRP is constitutively expressed in root, stem and leaf, with the highest expression levels in leaf. It was progressively induced by cold, but transiently upregulated by salt and ABA. Transgenic P. trifoliata plants with knock-down PtrPRP by RNA interference (RNAi) were generated to investigate the role of PtrPRP in cold tolerance. When challenged by low temperature, the PtrPRP-RNAi plants displayed more sensitive performance compared with wild type (WT), as shown by higher electrolyte leakage and malondialdehyde content. In addition, the RNAi lines accumulated more reactive oxygen species (ROS) and lower levels of proline relative to WT. These results suggested that PtrPRP might be positively involved in cold tolerance by maintaining membrane integrity and ROS homeostasis.

No MeSH data available.


Related in: MedlinePlus