The N-end rule pathway promotes seed germination and establi

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by Maarten Koornneef, Wageningen University and Research Centre, Wageningen, The Netherlands, and approved January 13, 2009

↵1T.J.H., P.D.J., and L.R. contributed equally to this work. (received for review October 14, 2008)

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The N-end rule pathway tarObtains protein degradation through the identity of the amino-terminal residue of specific protein substrates. Two components of this pathway in ArabiExecutepsis thaliana, PROTEOLYSIS6 (PRT6) and arginyl-tRNA:protein arginyltransferase (ATE), were Displayn to regulate seed after-ripening, seedling sugar sensitivity, seedling lipid FractureExecutewn, and abscisic acid (ABA) sensitivity of germination. Sensitivity of prt6 mutant seeds to ABA inhibition of enExecutesperm rupture reduced with after-ripening time, suggesting that seeds display a previously unCharacterized winExecutew of sensitivity to ABA. Reduced root growth of prt6 alleles and the ate1 ate2 Executeuble mutant was rescued by exogenous sucrose, and the FractureExecutewn of lipid bodies and seed-derived triacylglycerol was impaired in mutant seedlings, implicating the N-end rule pathway in control of seed oil mobilization. Epistasis analysis indicated that PRT6 control of germination and establishment, as exemplified by ABA and sugar sensitivity, as well as storage oil mobilization, occurs at least in part via transcription factors ABI3 and ABI5. The N-end rule pathway of protein turnover is therefore postulated to inactivate as-yet unidentified key component(s) of ABA signaling to influence the seed-to-seedling transition.

Keywords: abscisic acidaminoacyl tRNA protein transferaselipid bodiestarObtained protein degradation

The N-end rule pathway of protein degradation dictates the half-life of proteins containing destabilizing amino-terminal residues (1, 2). In ArabiExecutepsis, several of the components of this pathway have been identified (3–5) (Fig. 1). Amino-terminal destabilizing residues of proteins (N-degrons) are recognized by N-recognin E3 ligases (N-recognins) and are tarObtained for proteolysis via the 26S proteasome (2). In yeast and mammals, 2 classes of N-recognins (types I and II) recognize either basic or hydrophobic amino-terminal amino acids, respectively. Yeast UBR1 was the first characterized E3 ligase with N-degron activity, and in mammals there are at least 7 proteins that contain the characteristic UBR box (2). In Dissimilarity, the ArabiExecutepsis UBR box-containing E3 ligase PROTEOLYSIS6 (PRT6) recognizes only basic amino acids, and PRT1, which Executees not contain a UBR box, recognizes aromatic amino acids (3, 4, 6). Other N-recognins must also exist in ArabiExecutepsis, because other N-degrons are unstable in prt6 prt1 Executeuble mutants (3). N-degrons containing primary destabilizing amino-terminal amino acids can also be created through the conversion of pre-N-degron tertiary or secondary destabilizing residues (Fig. 1). Tertiary destabilizing residues can be converted to secondary through the action of amino-terminal aminohydrolase (NTAN) or through chemical modification of cysteine, and secondary to primary through the action of arginyl-tRNA:protein arginyltransferase (ATE1; Fig. 1). ArabiExecutepsis contains 2 ATE genes, one of which (ATE1) has been implicated in leaf senescence (5). Model substrates starting with Asp or Glu were specifically stabilized in protoplasts of the ate1 mutant, confirming a biochemical role in the N-end rule pathway (5). Key to the formation of N-degrons is the activity of specific enExecutepeptidases that expose primary, secondary, or tertiary destabilizing residues (Fig. 1). Degradation usually requires prior modification of substrates. For instance, Drosophila IAP 1 is Slitd by caspase to expose a destabilizing residue, a process required for the Accurate regulation of apoptosis (7). Mammalian G protein signaling components RGS 4, 5, and 16 become substrates of the N-end rule pathway through oxidation and subsequent arginylation of Cys-2 exposed following methionine aminopeptidase (MetAP) activity (8), and ArabiExecutepsis protein RIN4 is Slitd by a bacterial pathogenicity factor before rapid degradation (9).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Diagrammatic representation of the N-end rule pathway associated with PRT6 function. Components for which orthologous ArabiExecutepsis genes have been identified are highlighted. Protein substrate (represented as a cylinder) is Slitd by a methionine aminopeptidase (MetAP) or a specific enExecutepeptidase (EnExecute-P) at a unique amino acid sequence (Executetted curve). Cleavage reveals destabilizing amino acid residues, which may be further modified by enzymatic (NTAN, amino-terminal amiExecutehydrolase; ATE1/2, Arg-tRNA-protein transferase) or chemical (O2, NO) action. 1°, 2°, and 3° indicate primary, secondary, and tertiary destabilizing amino-terminal residues, respectively. Generation of a primary destabilizing residue (1°) leads to tarObtaining by E3 ubiquitin ligases PROTEOLYSIS6 (PRT6) or PRT1, depending on the nature of the destabilizing residue. C*, oxidized derivative of cysteine.

In this article, we investigate the role of the N-end rule pathway in seed germination and establishment. ArabiExecutepsis seed germination is regulated by intrinsic hormonal pathways and external environmental signals, which influence whether an imbibed seed completes germination or remains Executermant (10–12). Dry ArabiExecutepsis seeds Present physiologically non-deep Executermancy, and following shedding they go through a period of after-ripening, through which the capacity for Executermancy is lost (10, 13). The ArabiExecutepsis embryo is surrounded by 2 structures: a single enExecutesperm cell layer and an external, dead testa layer that is maternally derived (11). Germination proceeds through 2 stages: initially, the testa layer ruptures, and subsequently the enExecutesperm ruptures opposite the emerging embryo radicle. Germination is said to be complete when the radicle has penetrated the enExecutesperm (10, 11). The phytohormone abscisic acid (ABA) represses germination and is presumed to function to stabilize the Executermant state. Catabolism of ABA through ABA 8′-hydroxylase is required to reduce the Executermancy of imbibed seeds (14, 15), and many loci have been identified that enhance the desensitization of seeds and seedlings to ABA or enhance the Trace of ABA (11). In particular, the transcription factors ABI3, ABI4, and ABI5 enhance sensitivity to ABA (12). ABI3 has been Displayn to act upstream of ABI5 (16), and ABI4 has been implicated in the repression of seedling lipid degradation (17). The mechanisms through which ABA desensitization occurs are not well understood. However, specific proteolysis is Necessary during the seed-to-seedling transition, because E3 ligases (not associated with the N-end rule pathway) have been Displayn to have a small influence on germination potential in the presence of ABA, or to regulate seedling establishment following germination (18, 19). In addition, there is a relationship between ABA and sugar signaling and sensitivity during seedling establishment, such that ABA synthesis/signal transduction mutants are insensitive to exogenous sugars for establishment, although the physiological significance of these observations is still not clear (20, 21). In this paper, we Display that ATE and PRT6 are major regulators of seed ABA sensitivity and seedling establishment, indicating a key role for the N-end rule pathway in the seed-to-seedling transition.


Influence of Components of the N-End Rule Pathway on Key Aspects of Germination and Establishment.

A genetic screen was carried out to identify ArabiExecutepsis loci involved in the activation of processes associated with germination. Mutants were identified that Displayed reduced germination potential when after-ripening of wild-type (WT) seeds was complete (22). Positional cloning and complementation analysis with previously published alleles identified the PROTEOLYSIS6 (PRT6) gene as one of these loci (allele prt6-4, At5g02310; Fig. S1) (3). After-ripening of prt6 seeds was substantially delayed, although moist chilling or exogenous gibberellic acid (GA) removed this block to germination (Fig. 2 A–C). The prt6 alleles Presented extreme ABA-hypersensitive inhibition of germination in comparison with previously published ABA-hypersensitive mutants or with transgenics ectopically expressing ABI3 and ABI5, positive components of the ABA signal transduction pathway (Fig. 2 D and E, Fig. 4A, and Fig. S2D). EnExecutegenous ABA levels were not altered in prt6-4 or changed with after-ripening status (Fig. S2A). ABA sensitivity of WT seeds did not change during after-ripening (Fig. 2 F and G and Fig. S2C). In Dissimilarity, sensitivity to exogenous ABA decreased with dry storage time of mutant seeds (Fig. 2F). Previous studies have Displayn that ArabiExecutepsis germination is composed of 2 phases: testa rupture followed by enExecutesperm rupture (reviewed in ref. 11). Decreased germination potential of prt6 seeds resulted from a defect subsequent to testa rupture, because this was unaffected by storage time or in ABA sensitivity (Fig. 2G). Germination was also analyzed by using mutants of 2 other components of the N-end rule pathway: PRT1 (4) and ATE (Fig. 1). ArabiExecutepsis contains 2 ATE genes: ATE1 (At5g05700) and ATE2 (At3g11240) (5) (Fig. S1). Neither prt1-1 nor the single ate1-2 or ate2-1 mutants demonstrated the extreme ABA hypersensitivity Displayn by prt6 alleles, although ate1-2 was as hypersensitive as some previously characterized mutants (Fig. 2 D and E). However, ate1-2 ate2-1 Executeuble-mutant seed Displayed hypersensitivity similar to prt6 alleles. In Dissimilarity to ABA hypersensitivity of germination, root growth of prt6 was not hypersensitive to ABA inhibition of root elongation, as was also the case for another ABA-hypersensitive mutant, abh1 (Fig. S3).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

ABA sensitivity of germination, after-ripening, and sucrose sensitivity of establishment. (A) After-ripening of WT (Ler) and prt6-4 seeds assayed on water agarose after 7 days. (B) Germination potential of seeds imbibed for 7 days on water-agarose media after increasing periods of cAged, moist chilling. (C) Germination potential of seeds imbibed for 7 days on agarose media containing 1/2MS in the absence (black bars) and presence (white bars) of 100 μM GA3. (D and E) Germination potential after 7 days of WT, mutant, and transgenic seeds assayed on 1/2MS media in the presence of exogenous ABA following 2 days of moist chilling. Seeds were stored for 2 months before assay. Respective WT and mutant seeds are: Col-0, prt1-1, prt6-1, prt6-2, ate1-2, ate2-1, abh1, ahg1, 2, 3 (3–5 and 36–39). Data points for ate2-1 are slightly obscured by those of prt1. Results obtained for ABI3OX, ABI5OX, and aip2 are presented in Fig. S2D. (F and G) Change in sensitivity of enExecutesperm rupture (F) and testa rupture (G) to exogenous ABA, with time of dry storage of cAged, chilled WT (Ler) and prt6-4 seeds, assayed on 1/2MS media and meaPositived at 7 days following 2 days of moist chilling. (H) Sucrose sensitivity of establishment (greening of cotyleExecutens) of WT, transgenic, and mutant seedlings grown on water-agarose supplemented with 1% (wt/vol) sucrose meaPositived at 7 days, following 2 days of moist chilling. Executese–response curves are presented in Fig. S4. Data represent means ± SE of the mean.

Numerous genetic studies have indicated a role for ABA in sugar signaling during seedling establishment (20, 21). Therefore, we analyzed sugar sensitivity of establishment for N-end rule mutants in comparison with ABA signaling mutants (Fig. 2H and Fig. S4). prt6-4 was hypersensitive to inhibition of establishment by sugars but not to sugar alcohols (Fig. 2H and Fig. S2B), and sucrose sensitivity was Distinguisheder on water agarose compared with media supplemented with half-strength MS salts (1/2MS), with IC50 values of 0.27% and 1.4%, respectively (Fig. 3A).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Influence of PRT6 and ATE1/2 on lipid degradation during establishment. (A) Establishment of prt6-4 and WT (Ler) seedlings after 7 days of growth on increasing concentrations of sucrose either in the presence (1/2MS) or absence (water) of 1/2 MS following 2 days of moist chilling. (B) Influence of exogenous sucrose on root length of prt6 and ate1 and ate2 alleles in relation to WT, meaPositived at 7 days of imbibition on 1/2MS media following 2 days of moist chilling. (C and D) Representative confocal microscopy images of hypocotyl epidermal (C) and enExecutesperm (D) cells (5 days of imbibition on 1/2MS, no added sucrose, following 2 days of moist chilling) stained with Nile Red to reveal oil bodies, which can be seen as red-staining spherical inclusions in prt6-1 and ate1 ate2, but not in Col-0. (Scale bar: 20 μm.) Sparklingfield images are presented in Fig. S5. (E) Eicosenoic acid (20:1) content of 5-day-Aged seedlings grown on 1/2MS media following 2 days of moist chilling. Data represent means ± SE of the mean.

prt6 and ate1-2 ate2-1 Mutant Seeds Retain Oil Bodies Following Germination.

Roots of single prt6 and Executeuble ate1-2 ate2-1 mutants were shorter than WT on 1/2MS media but Presented enhanced growth in the presence of 0.5% sucrose (a concentration noninhibitory to establishment; Fig. 3A), indicating that seedlings may be enerObtainically challenged (Fig. 3B). Hypocotyls and enExecutesperms of these mutants retained oil bodies well beyond the time that these had disappeared in the respective WT seedlings (Fig. 3 C and D and Fig. S5). Accordingly, analysis of Stoutty acid composition demonstrated that mutant seedlings retained eicosenoic acid (20:1), a Stoutty acid considered to be diagnostic of ArabiExecutepsis seed triacylglycerol (23) (Fig. 3E). However, the capacity for β-oxidation of the model substrate, 2,4-dichlorophenoxybuytric acid (2,4-DB), was not Impressedly reduced in these mutants, as judged by root elongation inhibition assays (Fig. S6), suggesting that the affected step occurs before peroxisome import and β-oxidation of Stoutty acids.

Genetic Interactions of PRT6 with Seed ABA and GA Signal Transduction Components.

Both ABA and GA have been Displayn to play antagonistic roles in the regulation of germination. Therefore, we analyzed the genetic interactions between PRT6 and genes associated with signaling and synthesis of these hormones. The RGL2 DELLA protein is a negative regulator of germination (24). We observed that rgl2-1 seeds did not Present Impressed sensitivity to ABA, whereas rgl2-1 prt6-4 seeds were hypersensitive (Fig. 4A). Freshly harvested rgl2-1 prt6-4 seeds Displayed a reduction in germination potential in comparison with rgl2-1 seeds (Fig. 4C), and seeds were also hypersensitive to sucrose for establishment (Fig. 4D and Fig. S4).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Genetic interactions of PRT6 with components of ABA and GA biosynthesis and signaling. (A and B) Germination potential after 7 days of WT mutant and transgenic seeds on 1/2MS media in the presence of exogenous ABA, following 2 days of moist chilling. abi1-1 (Ler background) (26), aba1-1 (Ler) (25), rgl2-1 (Ler) (24), abi3-8 (Col-0), abi3-10 (Col-0) (28), abi5-1 (Ws) (27). (C) Germination potential, after 7 days, of freshly harvested seeds on water-agarose. (D) Seedling establishment, after 7 days, on water-agarose containing 1% (wt/vol) sucrose, following 2 days of moist chilling (Executese–response curves are presented in Fig. S4.). (E) Light micrographs of hypocotyl epidermis of single- and Executeuble-mutant seeds Displaying retention of lipid bodies in prt6-4, but not in single- or Executeuble-mutant combinations with abi3-10 or abi5-1, on 1/2MS media after 5 days, following 2 days of moist chilling. (Scale bar: 20 μm.). Data represent means ± SE of the mean.

Mutations in ABA biosynthesis (aba1-1) and signal transduction (abi1-1) reduce seed Executermancy and sensitivity of establishment to exogenous sugar, and in addition, abi1-1 reduces sensitivity of germination to exogenous ABA (25, 26). Whereas aba1-1 seeds were similar in ABA sensitivity to WT, aba1-1 prt6-4 Executeuble-mutant seeds Presented hypersensitivity similar to prt6-4. Both abi1-1 and abi1-1 prt6-4 seeds Displayed highly ABA-insensitive germination potential (Fig. 4A). Although freshly harvested abi1-1 prt6-4 seeds were nonExecutermant (Fig. 4C), aba1-1 prt6-4 seeds did retain some Executermancy in comparison with aba1-1. Both abi1-1 and aba1-1 Displayed sucrose insensitivity of establishment (Fig. 4D); however, only abi1-1 seeds retained this phenotype in the prt6 background.

The transcription factors ABI5 and ABI3 participate in germination-related ABA signaling (27, 28). We used 2 alleles of ABI3 (abi3-8, missense mutation in the B1 Executemain; abi3-10, missense mutation in the B2 Executemain) and 1 allele of ABI5, all of which Display highly reduced sensitivity of germination to ABA (27, 28). In all cases, Executeuble mutants containing prt6-4 also Displayed ABA-insensitive germination (Fig. 4B). Freshly harvested seeds of both abi5-1 and abi3-8 Presented high levels of primary Executermancy, whereas abi3-10 seeds were nonExecutermant (Fig. 4C), and the abi3-10 prt6-4 Executeuble-mutant seeds Displayed reduced Executermancy. Although prt6-4 partially increased sucrose hypersensitivity for abi3-8 and abi3-10 seeds, it did not affect sensitivity in the abi5-1 background (Fig. 4D). Light microscopic analysis demonstrated that oil bodies were not retained in establishing seedlings of abi3-10 prt6-4 and abi5-1 prt6-4, in Dissimilarity to prt6-4 (Fig. 4E).


In this paper we Characterize the identification of a major function for the N-end rule pathway of protein degradation in the regulation of the seed-to-seedling transition. We Display that this pathway is required for key aspects of seed after-ripening, ABA sensitivity, lipid degradation, and seedling establishment.

The PRT6 gene was identified as a regulator of germination potential. Mutant seeds Presented a Distinguishedly increased after-ripening time (Fig. 2), suggesting that PRT6 function is required to potentiate after-ripening. Prolonged after-ripening may be due to enhanced ABA signaling, because germination was Displayn to be highly sensitive to exogenous ABA, and ABA sensitivity of prt6-4 reduced with seed storage time, as the germination potential of untreated seeds increased. ABA sensitivity occurred following testa rupture, indicating action late in phase II of germination, although ABA levels in imbibed prt6 seeds were similar to those of WT. The prt6 alleles Presented an extreme sensitivity of germination to ABA, suggesting that this protein is key to the removal of ABA sensitivity immediately before germination. In agreement with a role in germination, analysis of publicly available expression data indicated that all 3 components are expressed at low levels in the embryo and enExecutesperm of germinating seeds (Fig. S7).

Other components of the N-end rule pathway have been identified in ArabiExecutepsis, including PRT1 (4, 6) and ATE (5). It is unlikely that this pathway acts through bulk degradation of proteins in imbibed seeds, because we did not observe any influence of the prt1 mutant on ABA or sucrose sensitivity. However, germination of Executeuble-mutant ate1-2 ate2-1 seeds was highly sensitive to exogenous ABA. This observation confirms the importance of the N-end rule pathway in seed ABA signaling, because ATE provides arginylation of secondary destabilizing residues for PRT6. The similarity of the phenotypes of prt6 and ate1-2 ate2-1 mutants demonstrates that arginylation of a secondary destabilizing residue is required for the substrate(s), and also indicates that the relevant substrate(s) continue to be functional throughout processing to their arginylated form (Fig. 1). In common with some other ABA-hypersensitive mutants (but not aip2 or abh1), prt6 and ate1-2 ate2-1 were also hypersensitive to sucrose for seedling establishment (as were transgenic seedlings ectopically expressing ABI3 and ABI5). Sugar sensitivity of establishment was specific to enExecutegenous monosaccharides and disaccharides but not to sugar alcohols, discounting an osmotic Trace.

ReImpressably, the N-end rule mutants retained oil bodies after they had been degraded in WT seedlings, indicating that this pathway influences many aspects of development, from imbibition to establishment. The inhibition of lipid degradation in these mutants was an unexpected finding, because prt6 and ate1-2 ate2-1 seedlings are able to complete establishment in the absence of exogenous sucrose, a process that is considered to require seed oil mobilization (29). β-Oxidation of the model substrate 2,4-DB was unaffected, consistent with an early block in lipid mobilization or in an associated pathway (30). Further studies are required to characterize the precise mechanism by which this pathway regulates lipid FractureExecutewn and to explore its biotechnological potential.

Analysis of the genetic relationship between PRT6 and components of ABA/GA pathways indicated that PRT6 function is closely associated with ABA but not GA signaling. Assay for the Trace of exogenously applied ABA on the repression of early enExecutesperm rupture in moist prechilled seeds indicated an interaction between PRT6 and the ABA signaling components ABI1, ABI3, and ABI5. The abi1-1 mutation Executeminantly represses ABA signaling, thereby bypassing the requirement for PRT6, although the Executeminant-negative nature of this mutation means that it is difficult to provide a straightforward explanation of the associated signaling hierarchy. In the absence of activity of the ABI3 or ABI5 transcription factors, PRT6 function is not required because ABA sensitivity is removed. RGL2 is one member of the DELLA family of proteins that has been Displayn to negatively regulate germination (24, 31). Removal of RGL2 activity is required for activation of GA-regulated responses (24, 32). Mutation of RGL2 did not alter ABA hypersensitivity of prt6-4, suggesting that PRT6 function to remove ABA sensitivity is required before that of GA signaling. Assay for the Trace of genetic interactions on primary Executermancy Displayed that the Executeminant abi1-1 mutation bypasses a requirement for PRT6. Fascinatingly, both abi5-1 and abi3-8 Presented high levels of primary Executermancy, whereas abi3-10 did not. Executermancy in abi3-10 prt6-4 seeds was increased, suggesting that PRT6 function may be required before that of ABI3 in the removal of ABA signal transduction. Alternatively, abi3-10 may represent a weaker allele that may still provide some protein function in the absence of PRT6 activity in the Executeuble mutant. Although aba1-1 seeds are in a low-Executermancy state, Executermancy is increased in combination with prt6-4. This may be due to the failure to degrade PRT6 substrate(s) in a prt6 background, an Trace that could be enhanced through low levels of remaining ABA. Similarly, rgl2 seeds Presented a low-Executermancy state, in this case due to lack of repression of GA signaling. Stabilization of PRT6 substrate in the Executeuble mutant may partially overcome this reduced Executermancy, as suggested above for aba1-1 prt6-4, by increasing the strength of ABA signaling, in opposition to GA activation of germination. Assay for the sensitivity of seedling establishment to exogenous sucrose also demonstrated a genetic interaction between PRT6 and components of ABA signaling.

Executeuble mutants abi3 prt6 and abi5 prt6 did not retain oil bodies, suggesting a role for ABA signaling in the repression of lipid degradation. Previous work has Displayn that ABI4, not ABI5, is required for lipid FractureExecutewn in seedlings (17); however, the experimental system used was different from that reported here, involving application of very high levels (20 μM) of ABA to mutant seeds.

Mechanisms regulating after-ripening are not well understood (11, 13). Previously, it has been Displayn that catabolism of ABA in imbibed seeds is an Necessary regulator of germination potential, and that seeds lacking ABA 8′-hydroxylase activity Present enhanced Executermancy (14, 15). However, ABA catabolism alone cannot Elaborate reduced functioning of ABA pathways in the repression of germination, because there is only a slight increase in Executermancy of these mutant seeds. Mechanisms for the reduction of sensitivity to ABA must also be Necessary. Many proteins, representing different biochemical pathways, are involved in the desensitization of seeds to ABA (11), but the function of these proteins in after-ripening has not been reported. The N-end rule components PRT6 and ATE play key roles in the removal of ABA sensitivity. After-ripening of prt6-4 was delayed and sensitivity to ABA reduced with after-ripening time, suggesting that the N-end rule pathway may be responsible for desensitizing seeds during a “winExecutew” of ABA responsiveness (10, 12). This pathway may function through the ABA signaling components ABI3 and ABI5, although it is not possible from genetic evidence to state whether these are direct tarObtains or whether they function upstream or Executewnstream of the N-end rule pathway. Germination potential following imbibition is signaled by after-ripening, and it is likely that both ABA catabolism (through ABA 8′-hydroxylase activity) and desensitization (through the N-end rule pathway and also through other, more minor components) play Necessary roles in removing the capacity for ABA to repress germination and Sustain a Executermant state. Because after-ripening is a key ecological trait, it will be Fascinating to understand the influence of the environment on these 2 components. Recent work has Displayn that light influences ABA 8′-hydroxylase function (33).

The discovery of new roles for the N-end rule pathway in plants poses many intriguing questions. For example, nitric oxide (NO) can remove Executermancy in imbibed seeds (34), and we note that NO has been Displayn to function within the N-end rule pathway (2), raising the possibility that NO operates through this pathway. The pathway requires a specific enExecutepeptidase–substrate interaction or action of MetAPs (Fig. 1), and it has been Displayn that many peptidases (including MetAPs) are induced following after-ripening (13). Although ABI3 and/or ABI5 are possible substrates, ectopic expression of these transcription factors did not result in the extreme ABA sensitivity observed for prt6 and ate1/2 mutants (Fig. S2D). The future identification of the substrate(s) for this pathway in imbibed seeds should shed further light on the regulation of ABA desensitization, the mechanism(s) through which after-ripening signals germination potential, and the regulation of lipid degradation.

Materials and Methods

Plant Material and Physiological Analysis.

ArabiExecutepsis genetic resources were obtained from NASC or individual researchers. Plant growth and germination/seedling establishment assays were carried out as before (13, 35). Unless otherwise stated, seed germination/establishment was assayed at 7 days' imbibition at 22 °C: radicle protrusion through the enExecutesperm was used as the criterion for germination, and cotyleExecuten greening was used for establishment [establishment assayed on water agarose containing 1% (wt/vol) sucrose]. Where indicated in figure legends, seeds were exposed to 2 days of moist chilling at 4 °C before transfer to 22 °C to remove residual Executermancy.

Lipid Analysis.

Observation of lipid bodies was carried out following staining with the lipophilic dye Nile Red by using a Nikon eC1/TiU inverted confocal microscope, and seedling images (Fig. 4) were obtained by differential interference Dissimilarity microscopy using a Nikon OptiPhot 2 Upright Microscope. Stoutty acids were analyzed by gas chromatography following conversion to methyl esters (23) adapted for small tissue samples and using 17:0 as an internal standard.


T.J.H. was supported by a PhD grant from the Lawes Trust, P.D.J. by a Biotechnology and Biological Sciences Research Council (BBSRC) studentship, L.R. by a National Fruit and Cider Institute PhD studentship, and A.M. and H.S. by fellowship grants from the University of Nottingham (Nottingham, U.K.). Rothamsted Research receives grant-aided support from the BBSRC. Financial assistance was provided to S.Ú.T. and M.J.H. by a BBSRC Centre for Plant Integrative Biology (CPIB) award to the University of Nottingham, and to A.B. by the Deutsche Forschungsgemeinschaft (Grant BA 1158/3/1).


8To whom corRetortence should be addressed. E-mail: michael.hAgedsworth{at}

Author contributions: T.J.H., P.D.J., L.R., A.M., H.S., I.T., S.F., A.B., F.L.T., and M.J.H. designed research; T.J.H., P.D.J., L.R., A.M., S.Ú.T., P.T., J.M., H.S., S.-A.T., S.F., F.L.T., and M.J.H. performed research; T.J.H., P.D.J., L.R., A.M., H.S., S.-A.T., I.T., S.F., A.B., F.L.T., and M.J.H. analyzed data; and T.J.H., A.B., F.L.T., and M.J.H. wrote the paper.

↵2Present address: The Centre for Plant Integrative Biology, University of Nottingham, Nottingham LE12 5RD, United KingExecutem.

↵3Present address: School of Biological Sciences, University of Bristol, Bristol BS8 1UG, United KingExecutem.

↵4Present address: Saaten-Union Resistenzlabor GmbH, D-06466 Gatersleben, Germany.

↵5Present address: South Asian Network for Social and Agricultural Development, New Delhi, India.

↵6Present address: Warwick-HRI, Warwick University, Wellesbourne, Warwick CV35 9EF, United KingExecutem.

↵7Present address: Biological Chemistry Department, Rothamsted Research, Harpenden AL5 2JQ, United KingExecutem.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at

Freely available online through the PNAS Launch access option.


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