Regulation of two germin-like protein genes during plum fruit development

Abstract

Germin-like proteins (GLPs) have several proposed roles in plant development and defence. Two novel genes (Ps-GLP1 and 2) encoding germin-like protein were isolated from plum (Prunus salicina). Their regulation was studied throughout fruit development and during ripening of early and late cultivars. These two genes exhibited similar expression patterns throughout the various stages of fruit development excluding two important stages, pit hardening (S2) and fruit ripening (S4). During fruit development until the ripening phase, the accumulation of both Ps-GLPs is related to the evolution of auxin. However, during the S2 stage only Ps-GLP1 is induced and this could putatively be in a H(2)O(2)-dependent manner. On the other hand, the diversity in the Ps-GLPs accumulation profile during the ripening process seems to be putatively due to the variability of endogenous auxin levels among the two plum cultivars, which consequently change the levels of autocatalytic ethylene available for the fruit to co-ordinate ripening. The effect of auxin on stimulating ethylene production and in regulating Ps-GLPs transcripts was also investigated. These data, supported by their localization in the extracellular matrix, suggest that auxin is somehow involved in the regulation of both transcripts throughout fruit development and ripening.

Fig. 1.

Phylogenetic relationships between P. salicina [Ps-GLP1 (EU310513), Ps-GLP2 (EU310512)]; P. persica [Pp-ABP19 (U79114), Pp-ABP20 (U81162)]; V. vinifera [Vv-GLP2 (ABH09468), Vv-GLP3 (AAQ63185), Vv-GLP6 (ABL60875)]; G. hirsutum [Gh-GLP1 (AAO92740)]; S. alba [Sa-GLP (P45854)]; A. thaliana [At-GLP3a (P94072), At-GLP5 (AAB51569), At-GLP6 (P92997)]; H. vulgare [Hv-GER1a (ABG46232), Hv-GER2a (ABG46233), Hv-GER3a (ABG46234), Hv-GER4d (ABG46236), Hv-GER5a (ABG46237)]; P. nil [Pn-GLP (P45853)]; A. lentiformis [Al-GLP (BAA78563)]; P. sativum [Ps-GER1 (CAB65369)]; N. plumbaginifolia [Np-NEC1 (Q9SPV5)]; B. unguiculata [Bu-GLP (BAC53790)]; P. patens [Pp-GLP3a (BAD86499), Pp-GLP6 (BAD86502)]; T. aestivum [Ta-GER2 (P15290), Ta-GER3 (P26759)]; L. perenne [Lp-OXO1 (CAC19429)]; and O. sativa [Os-GF2 (ABF98325)] based on full-length amino acid sequence. True germin, germin like protein (GLP) I, II, and III correspond to the different germin/GLP gene subfamilies.

Fig. 2.

Steady-state transcript levels of Ps-GLP1 and 2 assessed by QRT-PCR during flower and early fruit development of the ‘SH’ cultivar. The fertilized flowers stage is marked with an asterisk. The experiments were carried out in triplicate. The x-axis represents the developmental stages indicated by the number of days after bloom (DAB). Relative intensity in the y-axis refers to the fold difference in gene expression relative to ‘SH’ fruits 105 DAB for Ps-GLP1 and 2.

Fig. 3.

Steady-state transcript levels of Ps-GLP1 and 2 during Stage 1, Stage 2, and Stage 3 of ‘SH’ fruit development using the whole fruit (Stage 1 and Stage 2). However, during Stage 3 of fruit development the expression was determined in pulp (black bars) and in seeds (grey bars). The experiments were carried out in triplicate. The x-axis represents the developmental stage indicated by number of days after bloom (DAB) and by the name of stage. Other details are as described in Fig. 2.

Fig. 4.

Ethylene evolution and steady-state transcript levels of Ps-GLP1 and 2 during early ‘EG’ (left panel) and late ‘SH’ (right panel) plum fruit ripening. The expression was quantified in pulp (black bars) and in seeds (grey bars). The experiments were carried out in triplicate. The x-axis represents the developmental stage indicated by number of days after bloom (DAB). Other details are as described in Fig. 2.

Fig. 5.

(A) Ethylene production of ‘SH’ plum fruit treated with auxin (filled circles) 0 μM, (open squares) 1 μM, (open triangles) 10 μM, and (open diamonds) 100 μM. Fruit treated with 0 μM auxin served as the control. (B) The steady-state mRNA levels for Ps-GLP1 and 2 in ‘SH’ fruit during ripening at room temperature after treatment with 10 μM IAA. The x-axis in each figure represents days after auxin treatment. Other details are as described in Fig. 2.

Fig. 6.

Subcellular localization of Ps-GLP proteins fused to the GFP tag. Constructs consisting of either the control 35S::GFP, or 35S::GLP1-GFP or 35S::GLP2-GFP were used transiently to transform N. tabacum protoplasts. The subcellular localizations of the GFP protein under the control of 35S (A), the GLP1-GFP (B) or GLP2-GFP (C) fusion protein were analysed using confocal laser scanning microscopy. Light micrographs (centre panel) and fluorescence (left panel) images are merged (right panel) to illustrate the different locations of the two proteins. The length of the bar corresponds to 10 μm.