Arabidopsis seed development and germination is associated with temporally distinct metabolic switches

Abstract

While the metabolic networks in developing seeds during the period of reserve accumulation have been extensively characterized, much less is known about those present during seed desiccation and subsequent germination. Here we utilized metabolite profiling, in conjunction with selective mRNA and physiological profiling to characterize Arabidopsis (Arabidopsis thaliana) seeds throughout development and germination. Seed maturation was associated with a significant reduction of most sugars, organic acids, and amino acids, suggesting their efficient incorporation into storage reserves. The transition from reserve accumulation to seed desiccation was associated with a major metabolic switch, resulting in the accumulation of distinct sugars, organic acids, nitrogen-rich amino acids, and shikimate-derived metabolites. In contrast, seed vernalization was associated with a decrease in the content of several of the metabolic intermediates accumulated during seed desiccation, implying that these intermediates might support the metabolic reorganization needed for seed germination. Concomitantly, the levels of other metabolites significantly increased during vernalization and were boosted further during germination sensu stricto, implying their importance for germination and seedling establishment. The metabolic switches during seed maturation and germination were also associated with distinct patterns of expression of genes encoding metabolism-associated gene products, as determined by semiquantitative reverse transcription-polymerase chain reaction and analysis of publicly available microarray data. When taken together our results provide a comprehensive picture of the coordinated changes in primary metabolism that underlie seed development and germination in Arabidopsis. They furthermore imply that the metabolic preparation for germination and efficient seedling establishment initiates already during seed desiccation and continues by additional distinct metabolic switches during vernalization and early germination.

Figure 1.

Photosynthesis in developing Arabidopsis seeds. A, Morphology of developing seeds of Arabidopsis from 4 ± 1 to 20 ± 1 DAF. B, PSII fluorescence measurements (F_v/F_m; F₀ = 60 of approximately 100 seeds) of maturing seeds at 10 ± 1, 12 ± 1, 14 ± 1, and 18 ± 1 DAF. Values are average of two repeats. The average value for leaves from the same plant was 0.8 F_v/F_m (data not shown). C, Semiquantitative RT-PCR analyses of mRNAs encoding a light-harvesting complex protein of PSII (LHCII), PSI reaction center protein (PSI), and actin (ACT) as the control, in maturing seeds at 10 ± 1, 14 ± 1, and 17 ± 1 DAF.

Figure 2.

Changes in the contents of metabolites during the period of reserve accumulation in Arabidopsis seeds. Values of 14 ± 1 and 17 ± 1 DAF are normalized to the mean response calculated for 10 ± 1 DAF, which was given the value of 1. Value bars facing the right of each section indicate relative increased content as compared to 10 ± 1 DAF. Value bars facing the left of each section indicate the fold-decreased content relative to 10 ± 1 DAF. Values are representative of two independently grown sets of plants and are presented as the mean ± se of three biological repetitions of 30 mg of isolated seeds bulked from at least 10 plants for each time point. For statistical analysis and absolute content (nmol g fresh weight⁻¹) see Supplemental Tables S1 and S3. β-ala, Beta Ala; l-AsA, ascorbate; DHA, dehydroascorbate; Fru 6P, Fru 6 phosphate; Glu 6P, Glc 6 phosphate; Myo-ino, myo-inositol; 2-OG, 2-oxoglutarate; PhoA, phosphoric acid; ShikA, shikimate; ThrA, threonate; Treh, trehalose.

Figure 3.

Changes in the contents of metabolites during the desiccation period of Arabidopsis seeds. Values of mature seeds are normalized to the mean response calculated for 17 ± 1 DAF (representing the initiation of seed desiccation), which were given the value of 1. Values facing the right of each section indicate relative increased content as compared to 17 ± 1 DAF. Value bars facing the left of each section indicate the fold-decreased content relative to 17 ± 1 DAF. Values are representative of two independently grown sets of plants and are presented as the mean ± se of three biological repetitions of 30 mg of isolated seeds bulked from at least 10 plants for each time point. For statistical analysis and absolute content (nmol g fresh weight⁻¹) see Supplemental Tables S2 and S4. β-ala, Beta Ala; l-AsA, ascorbate; DHA, dehydroascorbate; Fru 6P, Fru 6 phosphate; Glu 6P, Glu 6 phosphate; Myo-ino, myo-inositol; 2-OG, 2-oxoglutarate; PhoA, phosphoric acid; ShikA, shikimate; ThrA, threonate; Treh, trehalose.

Figure 4.

PCA of metabolite profiles of distinct developmental stages during seed maturation. PCA is presented as the combinations of the first three dimensions, which together comprise 85.6% of the metabolite variance. Each data point represents an independent sample. The analysis of the data was performed using the TMEV software (Saeed et al., 2003). Component 1 explained 52.18% of the variance, component 2 explained 21.23%, and component 3 explained 12.17%. A to C represent components 1 + 2, 2 + 3, and 1 + 3, respectively. The combined percentages of the variance for each plot are given in brackets in each section.

Figure 5.

Semiquantitative RT-PCR analysis of mRNAs of selected genes in maturing and germinating Arabidopsis seeds. Total RNA was extracted independently from two different batches of isolated seeds at 10 ± 1, 14 ± 1, 17 ± 1 DAF, dry seeds, seeds after 72 h vernalization (imbibition for 72 h at 4°C in the dark), and seeds exposed to vernalization plus additional 24 h in light at 22°C (germination sensu stricto). At least three reactions were performed with each set of primers, as described in M&M. D, Dry seeds; V, vernalized seeds; G, germination sensu stricto; −RT, negative control with no RT reaction; PSI, PSI reaction center subunit III; FK, fructokinase; FB, Fru bisphosphate aldolase; GABATA, GABA trans aminase; ASPA, asparaginase; PKP, pyruvate kinase plastidic; PKC, pyruvate kinase cytosolic; SDH1, succinate dehydrogenase1; SDH2, succinate dehydrogenase2; ICL, isocitrate lyase; PEPCK, PEP carboxykinase; DHPS-2, dihydrodipicolinate synthase; THS, Thr synthase; TPP, trehalose-6-P phosphatase; ADH, alcohol dehydrogenase; CRU3-12S, storage protein; TBS2, Trp synthase β-subunit; FAH, fumarylacetoacetate hydrolase; ASN-1, Asn synthase; ACT, actin 2 (used as a control).

Figure 6.

Changes in the contents of metabolites during vernalization of Arabidopsis seeds. Values of seeds imbibed at 4°C in the dark for 72 h are normalized to the mean response calculated for dry seeds, which was given the value of 1. Value bars facing the right of each section indicate relative increased content as compared to dry seeds. Value bars facing the left of each section indicate the fold-decreased content relative to dry seeds. Values are representative of two independently grown sets of plants and are presented as the mean ± se of three biological repetitions of 30 mg of isolated seeds bulked from at least 10 plants for each time point. For statistical analysis and absolute content (nmol g fresh weight⁻¹) see Supplemental Tables S5 and S6. β-ala, beta Ala; l-AsA, ascorbate; DHA, dehydroascorbate; Fru 6P, Fru 6 phosphate; Glu 6P, Glc 6 phosphate; Myo-ino, myo-inositol; 2-OG, 2-oxoglutarate; PhoA, phosphoric acid; ShikA, shikimate; ThrA, threonate; Treh, trehalose.

Figure 7.

Changes in the contents of metabolites during the transition of Arabidopsis seeds from vernalization to germination sensu stricto. Values of seeds imbibed at 4°C in the dark for 72 h and then transferred to germinative conditions (21°C in the light) for additional 24 h are normalized to the mean response calculated for dry seeds. Value bars facing the right of each section indicate relative increased content as compared to dry seeds. Value bars facing the left of each section indicate the fold-decreased content relative to dry seeds. Values are representative of two independently grown sets of plants and are presented as the mean ± se of three biological repetitions of 30 mg of isolated seeds bulked from at least 10 plants for each time point. For statistical analysis and absolute content (nmol g fresh weight⁻¹) see Supplemental Tables S5 and S6. β-ala, beta Ala; l-AsA, ascorbate; DHA, dehydroascorbate; Fru 6P, Fru 6 phosphate; Glu 6P, Glc 6 phosphate; Myo-ino, myo-inositol; 2-OG, 2-oxoglutarate; PhoA, phosphoric acid; ShikA, shikimate; ThrA, threonate; Treh, trehalose.

Figure 8.

Schematic illustration of changes in the levels of metabolites synthesized by metabolic pathways localized to different subcellular organelles. A and B represent changes occurring during seed desiccation (transition from 17 ± 1 DAF to mature seeds; data derived from Fig. 3) and seed vernalization (transition from mature seeds to seeds vernalized for 72 h in the dark; data derived from Fig. 6), respectively. Blue and red letters indicate metabolites whose levels were either decreased or elevated, respectively. Metabolites whose levels were unchanged are marked by bold, black letters. Metabolites whose levels were not measured are marked in italics. Different organs are marked in green (plastids), pink (mitochondria), and purple (glyoxysomes). Arrows represent one or multiple enzymatic steps. AsA, Ascorbate; DHA, dehydroascorbate; FA, fatty acids; Fum, fumarate; Isocit, isocitrate; Mal, malate; 2OG, 2-oxoglutarate; OAA, oxaloacetate; 3PGA, 3-phosphoglycerate; Pyr, pyruvate; Shik, shikimate; Succ, succinate.