The Arabidopsis Aleurone Layer Responds to Nitric Oxide, Gibberellin, and Abscisic Acid and Is Sufficient and Necessary for Seed Dormancy1[C][W][OA] Paul C. Bethke2*, Igor G.L. Libourel2, Natsuyo Aoyama, Yong-Yoon Chung, David W. Still, and Russell L. Jones United States Department of Agriculture, Agricultural Research Service, Department of Horticulture, University of Wisconsin, Madison, Wisconsin 53706 (P.C.B.) Department of Plant and Microbial Biology, University of California, Berkeley, California 94720 (P.C.B., I.G.L.L., R.L.J.) Michigan State University, East Lansing, Michigan 48824 (I.G.L.L.) Department of Plant Sciences, California State Polytechnic University, Pomona, California 91768 (N.A., D.W.S.) and College of Life Sciences, Korea University, Seoul, Korea 136���152 (Y.-Y.C.) Seed dormancy is a common phase of the plant life cycle, and several parts of the seed can contribute to dormancy. Whole seeds, seeds lacking the testa, embryos, and isolated aleurone layers of Arabidopsis (Arabidopsis thaliana) were used in experiments designed to identify components of the Arabidopsis seed that contribute to seed dormancy and to learn more about how dormancy and germination are regulated in this species. The aleurone layer was found to be the primary determinant of seed dormancy. Embryos from dormant seeds, however, had a lesser growth potential than those from nondormant seeds. Arabidopsis aleurone cells were examined by light and electron microscopy, and cell ultrastructure was similar to that of cereal aleurone cells. Arabidopsis aleurone cells responded to nitric oxide (NO), gibberellin (GA), and abscisic acid, with NO being upstream of GA in a signaling pathway that leads to vacuolation of protein storage vacuoles and abscisic acid inhibiting vacuolation. Molecular changes that occurred in embryos and aleurone layers prior to germination were measured, and these data show that both the aleurone layer and the embryo expressed the NO-associated gene AtNOS1, but only the embryo expressed genes for the GA biosynthetic enzyme GA3 oxidase. The seeds of most angiosperms are dormant at maturity, and dormancy must be lost before germina- tion can occur (Bewley, 1997). This pause in the plant life cycle allows germination to occur under conditions favorable for growth of the seedling and in a season that provides sufficient time for completion of the next generation. Dormancy is a property of an intact seed, but several parts within the seed can contribute to seed dormancy (Bewley, 1997 Koornneef et al., 2000 Finch- Savage and Leubner-Metzger, 2006). In many cases, seed coverings, such as remnants of the fruit, the testa, and the endosperm, are significant barriers to embryo outgrowth (Groot and Karssen, 1987 Sanchez et al., 1990 Dahal et al., 1997 Debeaujon et al., 2000). This seed coat-imposed dormancy is widespread and more common than true embryo dormancy, where the em- bryo fails to initiate growth even when removed from the constraints imposed by the seed coverings (Bewley and Black, 1994 Ogawa et al., 2003). Seed coats are thought to restrain the growth of the embryo, and weakening of the seed coats, perhaps combined with an increase in the growth potential of the embryo axis, can result in radicle protrusion (Nabors and Lang, 1971). The contribution of seed coat weakening relative to increased embryo growth potential for dormancy loss, however, remains controversial (see discussion in Gong et al., 2005). Arabidopsis (Arabidopsis thaliana) is like many seeds in that both the embryo and seed coats have been implicated in the control of dormancy and germination (Bewley and Black, 1994 Debeaujon and Koornneef, 2000 Finch-Savage and Leubner-Metzger, 2006 Muller �� et al., 2006). Dormancy is genetically determined, and seeds with some genotypes are dormant after months or years of dry storage, whereas seeds with other genotypes lose dormancy within weeks (Koornneef et al., 2000). This process of dormancy loss can be hastened or slowed by environmental conditions. For example, a period of 1 This work was supported by the National Science Foundation (to R.L.J.), by the California Agricultural Research Initiative (to D.W.S.), by the Plant Signal Network Research Center of the Ministry of Science and Technology, and by the Biogreen 21 program of Rural Development Administration Republic of Korea. 2 These authors contributed equally to the paper. * Corresponding author e-mail pbethke@wisc.edu fax 608���262��� 4743. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Russell L. Jones (rjones@nature.berkeley.edu). [C] Some figures in this article are displayed in color online but in black and white in the print edition. [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a sub- scription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.093435 Plant Physiology, March 2007, Vol. 143, pp. 1173���1188, www.plantphysiol.org �� 2007 American Society of Plant Biologists 1173

cold and damp, referred to as stratification, often re- moves dormancy, but seeds may be more dormant when imbibed at temperatures of 28��C to 37��C (Bewley and Black, 1994). The molecular and biochemical parameters that un- derlie seed dormancy remain unknown despite a cen- tury of research in this area. Genetic evidence indicates strongly that abscisic acid (ABA) is central to the establishment and maintenance of seed dormancy (Hilhorst and Karssen, 1992 Jullien et al., 2000) and that gibberellin (GA) is important for germination (Debeaujon and Koornneef, 2000 Ogawa et al., 2003 Kucera et al., 2005). Recent data have shown that nitric oxide (NO) is a likely component of a signaling path- way that promotes a loss of dormancy (Bethke et al., 2004b, 2006b). Data from Arabidopsis suggest that NO might decrease the sensitivity of seeds to ABA (Bethke et al., 2006a), but a relationship between NO and GA signaling has not been established. Arabidopsis has been the subject of research on dormancy and germination for several decades. Arab- idopsis ecotypes vary widely in their depth of dor- mancy (Clerkx et al., 2004). Dormancy in dry seeds is lost after a few weeks at 25��C for seeds of the Colum- bia (Col) and Landsberg erecta (Ler) ecotypes, but several months are required for seeds of the C24 ecotype to become nondormant. Seeds of highly dor- mant ecotypes such as Cape Verde Islands (Cvi) and Kashmir-2 (Kas2) require almost 1 year to fully after ripen (Clerkx et al., 2004). The optimal germination temperature for Arabidopsis is approximately 15��C, and seeds imbibed at higher temperatures may have increased dormancy. The seed coats of Arabidopsis consist of the dead testa and a layer of living aleurone cells. The devel- opment and structure of the testa has been described in detail (Windsor et al., 2000 Debeaujon et al., 2003). The testa arises from cells of the inner and outer integuments of the ovule, and the outer cell layer of the outer integument produces mucilage and thickened cell walls, which give the outer surface of the seed its characteristic pattern (Windsor et al., 2000). Proantho- cyanadin pigments are deposited in the testa and contribute to Arabidopsis seed dormancy (Debeaujon et al., 2000, 2001, 2003). The living, single-cell-layered aleurone is the sole endosperm tissue, and it accumu- lates storage lipid and protein during seed maturation (Declercq et al., 1990 Penfield et al., 2004). A role for the Arabidopsis aleurone layer in supplying sugars to the growing seedling has been described (Penfield et al., 2004), and mutants with impaired lipid metab- olism in the aleurone layer and embryo have been identified that show defects in germination and early seedling growth (Eastmond et al., 2000 Footitt et al., 2002). The mobilization of stored lipid in the Arabi- dopsis aleurone layer requires GA and is not blocked by ABA (Penfield et al., 2004). Here we report on experiments designed to examine the contribution of the Arabidopsis embryo, aleurone layer, and testa to seed dormancy and to determine where in the seed NO is perceived. The data indicate that under conditions of high water potential, the aleurone layer is the most important determinant of seed dormancy. The data also show clearly that the aleurone layer is responsive to NO, as well as to GA and ABA, in ways that are consistent with the phys- iology of dormancy and germination in this species. RESULTS Arabidopsis Seeds Remain Dormant When the Testa Is Removed Mature Arabidopsis seeds contain an embryo en- veloped in an aleurone layer that is in turn surrounded by the testa. Previous work has shown convincingly that pigments in the testa contribute significantly to seed dormancy (Debeaujon et al., 2000). Removal of the testa from imbibed, dormant C24 Arabidopsis seeds, however, was not sufficient to remove seed dor- mancy as long as an intact aleurone layer was present. These testa-less seeds, such as those shown in Figure 1, A to C, could remain viable and dormant for over 1 month without germinating or greening when incu- bated on agarose in the light. Dormancy was lost when the aleurone layer was damaged or removed (see below). To demonstrate that testa-less seeds retained the ca- pacity to germinate, they were treated with vapors from a KCN solution. In a previous report, it was shown that KCN vapors effectively reduced Arabidopsis seed dor- mancy (Bethke et al., 2006b). Testa-less seeds treated with KCN vapors germinated 100% after 5 d, whereas testa-less seeds exposed to water vapor failed to ger- minate readily, and only 10% germinated within 5 d (Fig. 1D). Previous experiments with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1- oxyl-3-oxide (c-PTIO) suggested that NO participates in the loss of Arabidopsis seed dormancy resulting from KCN treatment (Bethke et al., 2006b). The germi- nation of testa-less seeds treated with KCN vapor was reduced by 100 mM c-PTIO, suggesting that NO plays a role in that process as well (Fig. 1D). We tested whether testa-less seeds responded to NO by exposing them to gaseous NO, as described previ- ously (Libourel et al., 2006). As shown in Figure 2A, over 90% of testa-less seeds germinated within 4 d of treatment with NO gas compared with less than 20% for similar testa-less seeds treated with air. The effect of NO gas on testa-less seeds was much more dramatic than the effect on intact seeds treated for 48 h with the same gas mixture (Fig. 2B). Whereas germination percentages 4 d after a 24-h treatment with NO were approximately 95% for testa-less seeds, they were only about 20% for intact seeds treated for 48 h (Fig. 2B). It was observed, however, that testa-less C24 seeds were less dormant than intact seeds, because about 15% of testa-less seeds germinated 4 d after being exposed to air for 24 h, whereas 0% of intact seeds exposed to air germinated at this time. These experiments with Bethke et al. 1174 Plant Physiol. Vol. 143, 2007

testa-less seeds demonstrate that the major determi- nant of seed dormancy resides in the aleurone layer and/or embryo and that either the aleurone layer or embryo was responsive to KCN and NO. The fact that testa-less seeds (Figs. 1D and 2A) are less dormant than intact seeds in the absence of KCN or NO treatment (Figs. 1D and 2B) confirms prior observa- tions showing that the testa contributes to dormancy in Arabidopsis (Debeaujon et al., 2000), perhaps by acting as a barrier against passage of small molecules. Embryos Removed from Dormant Arabidopsis Seeds Are Not Dormant To differentiate between components of seed dor- mancy that originate in the embryo and those that originate in the aleurone layer, the responses of iso- lated embryos and isolated seed coats were studied. Seed coats consisted of the living aleurone layer and the dead, adhering testa, and throughout this article we refer to these as Arabidopsis aleurone layers. This terminology is consistent with that used for cereal aleurone layers, which are the aleurone layer with adhering testa and pericarp. Figure 3 shows photographs of embryos that were removed from several ecotypes of Arabidopsis seeds within hours of imbibition and then incubated on agarose. The Col seeds used for this experiment were fully after ripened and showed no dormancy. The C24, Cvi, and Kas2 ecotype seeds were dormant and had final germination percentages of less than 5%. Regard- less of the dormancy status of the seed, however, none of these seeds had true embryo dormancy. All isolated embryos grew and greened within 3 to 4 d of removal from the seed coats. Indeed, under the conditions of our experiment, embryos from highly dormant Cvi and Kas2 ecotypes grew more vigorously than em- bryos from nondormant Col seeds (Fig. 3). We compared the ability of embryos from dormant and nondormant C24 seeds to grow on agarose con- taining mannitol or polyethylene glycol (PEG) at con- centrations up to 500 mOsmol. As expected, higher osmotic potentials slowed the rate of embryo growth, and this is shown in the photographs of embryos grown on mannitol in Figure 4A. Embryos from dor- mant seeds failed to grow at 400 mOsmol mannitol, while embryos from nondormant seeds were able to grow at 500 mOsmol mannitol (Fig. 4A). Embryo growth was quantified by measuring changes in sur- face area, and these data are plotted in Figure 4B for the embryo as a whole, and in Figure 4C for the cotyledons, hypocotyls, and roots individually. With Figure 1. Arabidopsis seeds remain dormant when the testa is removed but germinate when exposed to cyanide vapors. Seeds lacking the testa are shown in A to C 3 d (A) and 28 d (B and C) after testa removal. The seed in C was exposed to KCN vapors for 3 d beginning 25 d after testa removal. D, Germination percentages after 5 d for intact seeds exposed to water vapor or KCN vapor for 2 d, and for seeds with the testa removed exposed to water vapor, KCN vapor, or KCN vapors in the presence of c-PTIO. Each experiment was repeated at least three times with five to 14 seeds per treatment. Bars with different lowercase letters are significantly different with P , 0.05. Figure 2. Germination of Arabidopsis seeds lacking the testa is stim- ulated by NO gas. Purified NO gas or air were passed over seeds with the testa removed for 1 d (A) or over intact seeds for 2 d (B), and germination was scored every day for 4 d. Data are means 6 SE for six to 17 seeds. Arabidopsis Aleurone Layer and Seed Dormancy Plant Physiol. Vol. 143, 2007 1175