WRI1 is required for seed germination and seedling establishment

Abstract

Storage compound accumulation during seed development prepares the next generation of plants for survival. Therefore, processes involved in the regulation and synthesis of storage compound accumulation during seed development bear relevance to germination and seedling establishment. The wrinkled1 (wri1) mutant of Arabidopsis (Arabidopsis thaliana) is impaired in seed oil accumulation. The WRI1 gene encodes an APETALA2/ethylene-responsive element-binding protein transcription factor involved in the control of metabolism, particularly glycolysis, in the developing seeds. Here we investigate the role of this regulatory factor in seed germination and seedling establishment by comparing the wri1-1 mutant, transgenic lines expressing the WRI1 wild-type cDNA in the wri1-1 mutant background, and the wild type. Plants altered in the expression of the WRI1 gene showed different germination responses to the growth factor abscisic acid (ABA), sugars, and fatty acids provided in the medium. Germination of the mutant was more sensitive to ABA, sugars, and osmolites, an effect that was alleviated by increased WRI1 expression in transgenic lines. The expression of ABA-responsive genes AtEM6 and ABA-insensitive 3 (ABI3) was increased in the wri1-1 mutant. Double-mutant analysis between abi3-3 and wri1-1 suggested that WRI1 and ABI3, a transcription factor mediating ABA responses in seeds, act in parallel pathways. Addition of 2-deoxyglucose inhibited seed germination, but did so less in lines overexpressing WRI1. Seedling establishment was decreased in the wri1-1 mutant but could be alleviated by sucrose. Apart from a possible signaling role in germination, sugars in the medium were required as building blocks and energy supply during wri1-1 seedling establishment.

Figure 1.

Increased sensitivity of wri1-1 seed germination to ABA. Seed germination of wild type (squares), line 101 expressing the WRI1 cDNA under the control of the 35S-CaMV promoter in the wri1-1 background (triangles), and wri1-1 (circles) was analyzed. Plants were grown in the absence (top section) and in the presence (bottom section) of 50 mm Suc. Germination is reported as fraction relative to the zero control. Germination was scored by radicle emergence. Four independent sets of data were averaged per treatment and sd is indicated.

Figure 2.

Impairment of wri1-1 germination in the presence of osmolites. Sodium chloride (NaCl; A), sorbitol (Srb; B), Glc (C), and Suc (D) were added to the medium at the concentrations indicated. The fraction of germination in the presence of osmolite is presented relative to germination without osmolite. Error bars represent sd of the mean of three independent assays. Plant lines and symbols are as described in Figure legend 1 and shown here in A.

Figure 3.

Expression of ABA-responsive genes in wri1-1. Transcript analysis was conducted following transfer of seedlings to medium containing 50 μm ABA, 50 mm Suc, or lacking these compounds as indicated by (−) or (+), respectively. Seedlings were grown on medium with or without Suc for 6 d and then transferred to the same medium containing or lacking ABA. RNA was extracted after three additional days. The RNA was blotted and the blots probed with the indicated gene. The rRNA bands from the ethidium bromide-stained gel are shown as a loading control.

Figure 4.

Interaction of ABI3 and WRI1. A, Germination assays on early desiccation seeds of wild type (black bars), abi3-3 (densely hatched bars), abi3-3 wri1-1 (sparsely hatched bars), and wri1-1 (white bars) are shown. ABA was present (+) at 10 μm or absent (−). Absolute percent germination is reported as the average of the mean of three independent experiments with the error bars representing sd. B, mRNA abundance of WRI1 and ABI3 in dry seeds (D), 16 h (16), and 48 h (48) after imbibition. The RNA was blotted and the blots probed with the indicated gene. The rRNA bands from the ethidium bromide-stained gel are shown as a loading control.

Figure 5.

Seed germination in the presence of sugar analogs. Germination is reported as fraction relative to the zero control. Glc (Glc; A), 2-deoxyglcuose (2-d-Glc; B), and 3-O-methylGlc (3-M-Glc; C) were added to the medium at concentrations as indicated. Black bars represent the wild type, densely hatched bars line 101, sparsely hatched bars line 106, which is similar to line 101 but a stronger WRI1 cDNA expresser, and white bars represent wri1-1. All assays were performed in triplicate with sd of the mean represented by the error bars.

Figure 6.

Glycolytic enzyme activities and mRNA abundance during germination and early seedling establishment. A, Enzyme activities for hexokinase represented as glucokinase or fructokinase activities and pyruvate kinase. Plants were grown on agar with half-strength Murashige and Skoog medium but lacking Suc. Tissues were harvested at times after incubation as indicated. The black bars represent wild type, densely hatched bars line 101, sparsely hatched bars line 106, and white bars the wri1-1 mutant. Error bars depict sd of the mean of enzyme activity obtained for three different protein preparations. B, mRNA abundance of HXK1 and PK1. The blot is identical to the blot shown in Figure 4B, but was stripped and reprobed with the gene-specific probes as indicated. The rRNA bands from the ethidium bromide-stained gel are shown as a loading control.

Figure 7.

Impaired seedling establishment in the wri1-1 mutant. A, Appearance of 12-d-old seedlings of wild type and wri1-1 grown in the presence (+) or absence (−) of 50 mm Suc. B, Root growth on medium lacking Suc. Symbols are as shown in the figure and as described in the Figure 1 legend with the addition of an inverted triangle for line 106. The error bars represent sd of the mean of at least 15 root length measurements.

Figure 8.

Light micrographs of cross sections of wild-type and wri1-1 roots. The plants were grown in the presence of 50 mm Suc. The sections were derived from the differentiation zone of the roots. The size bar represents 200 μm. CC, Central cylinder; E, endodermis; RH, root hair.