ABSCISIC ACID INSENSITIVE3 Is Involved in Cold Response and Freezing Tolerance Regulation in Physcomitrella patens

Abstract

Synopsis This work demonstrates that PpABI3 contributes to freezing tolerance regulation in Physcomitrella patens. Transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3) is known to play a major role in regulating seed dormancy, germination, seedling development as well as stress responses. ABI3 is conserved among land plants; however, its roles in non-seed plants under stress conditions have not been well characterized. In this study, we report that ABI3 is involved in freezing tolerance regulation during cold acclimation at least in part through ABA signaling pathway in moss Physcomitrella patens (P. patens). Deletion of PpABI3 (Δabi3-1) compromises the induction of genes related to cold response and antioxidative protection, resulting in reduced accumulation of cryoprotectants and antioxidants. In addition, photosystem II (PSII) activity is repressed in Δabi3-1 during cold acclimation partially due to alternations of photosynthetic protein complexes compositions. The gametophyte of Δabi3-1 displays severe growth inhibition and developmental deficiency under low temperature condition, while two independent complementary lines display phenotypes similar to that of wild-type P. patens (WT). Furthermore, the freezing tolerance of Δabi3-1 was significantly affected by deletion of PpABI3. These data revealed that PpABI3 plays an important role in low temperature response and freezing tolerance in P. patens.

Keywords: ABA; ABI3; Physcomitrella patens; cold acclimation; freezing tolerance; photosynthetic protein complexes.

Figure 1

Deletion of PpABI3 inhibits growth of P. patens under cold condition. (A) Analysis of gametophyte growth. Seven-day-old tissues of WT, Δabi3-1 and two complementary lines were inoculated on fresh BCD medium, and then grown under growth temperature (25°C) or lower temperature (10°C) for 4 weeks before photographs were taken. (B) Representative images of gametophores of each strains. Gametophores of wild type, Δabi3-1and two complementary lines incubated under 25°C or 10°C were collected to show their different phenotypes. Bar = 2 mm for all panels. (C) Analysis of biomass increment in (A). Fresh weight of each strain was determined for biomass increment analysis in relation to the starting inoculated biomass. Error bars represent SD (n = 3) and Two-way ANOVA was used to determine the statistical significance (^**P < 0.01). (D) Number of gametophores that emerged on the colonies of each strain. The colonies were incubated under 25°C or 10°C for 4 weeks and gametophores that emerged were counted. Error bars indicate mean ± SD (n = 6) and Two-way ANOVA was used to determine the statistical significance (^**P < 0.01). (E) Number of leaves that generated per gametophore of each strain. The colonies were incubated under 25°C or 10°C for 4 weeks and representative gametophores were collected for estimation of the leaves amount. Error bars indicate mean ± SD (n = 10) and Two-way ANOVA was used to determine the statistical significance (^**P < 0.01). (F) Mean height of gametophores analysis for the wild type, Δabi3-1 and PpABI3ABC:Δabi3-1 lines. Error bars indicate mean ± SD (n > 60) and Two-way ANOVA was used to determine the statistical significance (^**P < 0.01).

Figure 2

PpABI3 participates in low temperature response of P. patens. (A–C) Analysis of cell injury and oxidative damage. Tissues of WT and Δabi3-1 with or without cold acclimation under 10°C were collected to determine electrolyte leakage (EL) rate (A), hydrogen peroxide (H₂O₂) content (B) and malonyldialdehyde (MDA) content (C). (D–F) Analysis the accumulation of cryoprotectants. Tissues of WT and Δabi3-1 with or without cold acclimation were collected to estimate proline content (D), total soluble sugar content (E) and total soluble protein content (F). Error bars represent SD (n = 3) and Two-way ANOVA was used to determine the statistical significance (^**P < 0.01). (G,H) Quantitative RT-PCR to analyse the expression of a late embryogenesis abundant (LEA)-like protein-coding gene (Pp1s267_21V6.1) and a sucrose synthase-coding gene (Pp1s93_98V6.1). PpACTIN5 (Pp1S381_21V6) was used as internal control. Error bars represent SD (n = 3) and Two-way ANOVA was used to determine the statistical significance (^**P < 0.01; ^***P < 0.001).

Figure 3

PpABI3 is involved in regulating enzymatic antioxidative signaling. (A–D) Antioxidant enzymatic activities analysis. Protonema after incubation under standard growth temperature (25°C) or low temperature (10°C) for 2 weeks were harvested to analyze ascorbate peroxidase (APX) activities (A), catalase (CAT) activities (B), glutathione peroxidase (POD) activities (C) and superoxide dismutase (SOD) (D). Error bars represent SD (n = 3) and two-way ANOVA was used to determine the statistical significance (^**P < 0.01). Experimental details are found in Materials and Methods.

Figure 4

Expression of antioxidant enzymes mediated by PpABI3 during cold acclimation. (A–D) The transcriptional levels of genes encoding ascorbate peroxidase (APX: Pp1S277_34V6), catalase (CAT: Pp1S422_8V6), glutathione peroxidase (POD: Pp1S98_2V6) and superoxide dismutase (SOD: Pp1S22_320V6) were analyzed by quantitative RT-PCR in wild-type and Δabi3-1 with or without cold acclimation. Error bars represent SD (n = 3), and two-way ANOVA was used to determine the statistical significance (^**P < 0.01; ^***P < 0.001).

Figure 5

Cold-responsive genes are regulated by PpABI3 during cold acclimation. (A) Relative transcription of three cold responsive genes. The transcriptional levels of three genes: PpCOR47 (Dehydrin-coding gene: Pp1s442_22V6.2), PpRD29A (Desiccation-responsive protein 29A: PP1S203_40V6.1) and PpCSP3 (Cold shock protein: Pp1s103_65V6.1) were analyzed by quantitative RT-PCR. (B) Relative transcription of representative transcription factors. The transcriptional levels of three cold responsive transcription factors: DREBP1/CBF (DRE/CRT binding factor: Pp1s60_228V6.1), AP2/EREBP (APETALA2/ethylene-responsive element binding protein: Pp1s373_18V6.1) and ABI5 (ABSCISIC ACID INSENSITIVE5: Pp1s49_161V6) were analyzed by quantitative RT-PCR. Error bars represent SD (n = 3), and two-way ANOVA was used to determine the statistical significance (^*P < 0.05; ^**P < 0.01; ^***P < 0.001).

Figure 6

Expression pattern of photosynthetic genes. (A–F) The transcriptional levels of genes related to photosystem manganese-stabilizing including PpPsaA (PhpapaCp039), PpPsaB (PhpapaCp040), PpPsbA (PhpapaCp046), PpPsbD (PhpapaCp044), PpPsbO (Pp1s60_65V6.1) and PpPsbP (Pp1s63_71V6.1) were determined by quantitative RT-PCR assay. Error bars represent SD (n = 3), and two-way ANOVA was used to determine the statistical significance (^*P < 0.05; ^**P < 0.01).

Figure 7

Deletion of PpABI3 reduces PSII activity during cold acclimation. (A–H) Chlorophyll fluorescence analysis. Two-week-old protonema of WT and Δabi3-1 with or with cold acclimation were collected to determine the maximum photochemical efficiency of PSII (F_v/F_m) (A), steady-state non-photochemical quenching (NPQ) (B), light-response curves of non-photochemical quenching (NPQ) (C,D), the efficiency of PSII quantum yield (ΦPSII) (E,F) and PSII electron transport rate (ETR) (G,H). Error bars represent SD (n = 3), two-way ANOVA was used to determine the statistical significance (^*P < 0.05; ^**P < 0.01). Experimental details are found in the Materials and Methods.

Figure 8

Deletion of PpABI3 affects assembly of PSII during cold acclimation. (A) Blue Native (BN)-gel electrophoresis analysis of thylakoid membrane protein complexes. Thylakoid membranes were isolated from WT and Δabi3-1 with or without cold acclimation, then solubilized with 2% DM and separated by BN-PAGE. A sample with an equal amount of chlorophyll (25 μg) was loaded in each lane. The BN gel (left) was further stained with Coomassie Brilliant blue (CBB; right). (B) Two-dimensional (2D) separation [BN-gel follows SDS-PAGE] of thylakoid protein complexes. Complexes in BN-gel were subsequently separated by SDS-urea-PAGE and stained with CBB to show their constituent subunits. Protein distribution differences between WT and Δabi3-1 were indicated by triangle symbol (black, standard condition; white, after cold acclimation).

Figure 9

PpABI3 is involved in ABA-dependent freezing tolerance regulation. (A) Electrolyte leakage rate analysis. Tissues of WT and Δabi3-1 with or without cold acclimation were treated or not with 1 μM ABA, then frozen to −4°C. After thawing, electrolyte leakage was measured to estimate cell injury. Error bars represent SD (n = 3), and two-way ANOVA was used to determine the statistical significance (^*P < 0.05; ^**P < 0.01; ^***P < 0.001). (B) Cell death rate estimation. Frozen-thawed cells were stained with 0.5% Evans Blue to analysis the cell death of WT and Δabi3-1. (C) Survival rate analysis. Tissues of WT and Δabi3-1 with or without cold acclimation were treated or not with 1 μM ABA, then frozen to −4°C, after thawing the cells survival rate were estimated according to Evans Blue staining in (B). Error bars represent SD (n = 3), and two-way ANOVA was used to determine the statistical significance (^***P < 0.001). (D) Recovery growing after frozen to −4°C. Frozen-thawed protonematal tissues of WT and Δabi3-1 were transferred onto fresh BCD medium and regrown under standard condition for 7 days to estimate the recovery growth capacity.