- © 2013 American Society of Plant Biologists. All rights reserved.
Abstract
Seed size in higher plants is determined by the coordinated growth of the embryo, endosperm, and maternal tissue. Several factors that act maternally to regulate seed size have been identified, such as AUXIN RESPONSE FACTOR2, APETALA2, KLUH, and DA1, but the genetic and molecular mechanisms of these factors in seed size control are almost totally unknown. We previously demonstrated that the ubiquitin receptor DA1 acts synergistically with the E3 ubiquitin ligase ENHANCER1 OF DA1 (EOD1)/BIG BROTHER to regulate the final size of seeds in Arabidopsis thaliana. Here, we describe another RING-type protein with E3 ubiquitin ligase activity, encoded by DA2, which regulates seed size by restricting cell proliferation in the maternal integuments of developing seeds. The da2-1 mutant forms large seeds, while overexpression of DA2 decreases seed size of wild-type plants. Overexpression of rice (Oryza sativa) GRAIN WIDTH AND WEIGHT2, a homolog of DA2, restricts seed growth in Arabidopsis. Genetic analyses show that DA2 functions synergistically with DA1 to regulate seed size, but does so independently of EOD1. Further results reveal that DA2 interacts physically with DA1 in vitro and in vivo. Therefore, our findings define the genetic and molecular mechanisms of three ubiquitin-related proteins DA1, DA2, and EOD1 in seed size control and indicate that they are promising targets for crop improvement.
INTRODUCTION
Seed size in higher plants is central to many aspects of evolutionary fitness and is also an important trait for agricultural purposes (Alonso-Blanco et al., 1999; Gómez, 2004; Orsi and Tanksley, 2009). For example, the seedlings of large-seeded plants are better able to withstand resource restrictions and abiotic stresses, whereas small-seeded plants are thought to have superior colonization abilities because they produce large numbers of seeds (Westoby et al., 2002; Moles et al., 2005). In agriculture, seed size is one of the most important components of seed yield. Crop plants (e.g., rice [Oryza sativa] and wheat [Triticum aestivum]) have undergone selection for larger seed size during domestication (Fan et al., 2006; Song et al., 2007; Gegas et al., 2010). However, the genetic and molecular mechanisms of seed size control are poorly understood.
In flowering plants, the seed comprises three major anatomical components, the embryo, the endosperm, and the seed coat, each with different genetic compositions. Seed development involves a double fertilization process in which one sperm nucleus fuses with the egg cell to give rise to the diploid embryo, while the other sperm nucleus fuses with the central cell to produce the triploid endosperm (Lopes and Larkins, 1993). By contrast, the seed coat that surrounds the embryo and endosperm is typically of maternal origin. Therefore, the size of a seed is regulated by the coordinated growth of maternal sporophytic and zygotic tissues. Several factors that influence seed size by the zygotic tissues have been recently identified in Arabidopsis thaliana, including HAIKU1 (IKU1), IKU2, MINISEED3 (MINI3), and SHORT HYPOCOTYL UNDER BLUE1 (SHB1) (Garcia et al., 2003; Luo et al., 2005; Zhou et al., 2009; Wang et al., 2010). IKU1, IKU2, and MINI3 function in the same pathway of seed development (Garcia et al., 2003; Luo et al., 2005; Wang et al., 2010). Downregulation of these three genes results in precocious cellularization of the endosperm and subsequently decreased seed size at maturity (Garcia et al., 2003; Luo et al., 2005; Wang et al., 2010). SHB1 promotes endosperm growth in the early phase of seed development by binding MINI3 and IKU2 promoters (Zhou et al., 2009). Seed size is also strongly affected by maternal tissues. Several factors that act in maternal sporophytic tissues to regulate seed size have been described in Arabidopsis, such as TRANSPARENT TESTA GLABRA2 (TTG2), APETALA2 (AP2), AUXIN RESPONSE FACTOR2 (ARF2)/MEGAINTEGUMENTA (MNT), KLUH/CYTOCHROME P450 78A5 (KLU/CYP78A5), and ENHANCER3 OF DA1 (EOD3)/CYP78A6 (Garcia et al., 2005; Jofuku et al., 2005; Ohto et al., 2005, 2009; Schruff et al., 2006; Adamski et al., 2009; Fang et al., 2012). TTG2 and EOD3/CYP78A6 affect seed growth by increasing cell expansion in the integuments (Garcia et al., 2005; Ohto et al., 2009; Fang et al., 2012), while AP2 may influence seed growth by limiting cell expansion in the integuments (Jofuku et al., 2005; Ohto et al., 2005, 2009). By contrast, KLU/CYP78A5 promotes seed growth by increasing cell proliferation in the integuments of ovules (Adamski et al., 2009), whereas ARF2/MNT restricts seed size by decreasing cell proliferation in the integuments (Schruff et al., 2006; Li et al., 2008). Therefore, the proliferation of integument cells to form the seed coat plays a critical role in setting seed size.
Several factors involved in ubiquitin-related activities have been known to influence seed size. A growth-restricting factor, DA1, is a ubiquitin receptor containing two ubiquitin interaction motifs (UIMs) that bind ubiquitin in vitro, and the da1-1 mutant forms large seeds as a result of changes in the maternal integuments of ovules (Li et al., 2008). Mutations in an enhancer of da1-1 (EOD1), which encodes the E3 ubiquitin ligase BIG BROTHER (BB; Disch et al., 2006; Li et al., 2008), synergistically enhance the seed size phenotype of da1-1, indicating that DA1 acts synergistically with EOD1/BB to regulate seed size. In rice, a quantitative trait locus for GRAIN WIDTH AND WEIGHT2 (GW2), encoding an E3 ubiquitin ligase, regulates grain size by restricting cell division (Song et al., 2007). An uncharacterized protein encoded by rice QTL FOR SEED WIDTH ON CHROMOSOME5/GW5 is required to limit grain size in rice (Shomura et al., 2008; Weng et al., 2008). GW5 physically interacts with polyubiquitin in a yeast two-hybrid assay, suggesting that GW5 may be involved in the ubiquitin-proteasome pathway (Weng et al., 2008). However, it is not clear whether these two factors act in maternal and/or zygotic tissues in rice.
To understand the molecular mechanisms of seed size control, we previously identified da mutants with large seeds in Arabidopsis (Li et al., 2008). Here, we describe Arabidopsis DA2, encoding a Really Interesting New Gene (RING)-type protein with E3 ubiquitin ligase activity, which acts maternally to restrict seed growth. Genetic analyses show that DA2 functions synergistically with the ubiquitin receptor DA1 to regulate seed size, but does so independently of the E3 ubiquitin ligase EOD1. We further demonstrate that DA1 interacts physically with DA2 in vitro and in vivo. Overexpression of rice GW2, a homolog of DA2, restricts seed and organ growth in Arabidopsis, suggesting a possible conserved function in Arabidopsis and rice. Thus, our findings define the genetic and molecular mechanisms of three ubiquitin-related proteins, DA1, DA2, and EOD1, in seed size regulation and suggest that combining the effects of DA1, DA2, and EOD1 from different seed crops provides a promising strategy for engineering crops with large seeds.
RESULTS
The da2-1 Mutant Produces Large Seeds
We previously identified a ubiquitin receptor, DA1, and an E3 ubiquitin ligase, EOD1/BB, as important factors influencing seed size in Arabidopsis (Li et al., 2008), suggesting that the ubiquitin pathway plays an important part in seed growth. To further understand the mechanisms of ubiquitin-mediated regulation of seed size, we collected the publicly available T-DNA insertion lines of some predicted ubiquitin ligase genes that were expressed in Arabidopsis ovules and/or seeds in several microarray studies (http://affymetrix.Arabidopsis.info/narrays/digitalnorthern.pl) and investigated their seed growth phenotypes. From this screen, we identified several T-DNA insertion mutants with altered seed size. We designated one of these mutants da2-1, referring to the order of discovery for large seed size mutants (DA means “large” in Chinese). Seeds produced by da2-1 were larger and heavier than the wild-type seeds (Figures 1A, 2A, 2C, and 2D). Seed number per silique and seed yield per plant in da2-1 were slightly higher than those in the wild type (Figures 1B and 1C). By contrast, the total number of seeds per plant in da2-1 was not significantly increased, compared with that in the wild type (Figure 1D). The da2-1 plants were higher than wild-type plants at the mature stage (Figure 1E). In addition, da2-1 mutant plants formed large flowers and leaves as well as increased biomass compared with wild-type plants (Figures 1F and 1G; see Supplemental Figures 1A to 1C online). The increased size of da2-1 mutant petals and leaves was not caused by larger cells (see Supplemental Figures 1D and 1E online), indicating that it is the number of petal and leaf cells that is higher.
Seed and Organ Size in the da2-1 Mutant.
(A) Projective area of Col-0, da2-1, and 35S:DA2#1 seeds. The seeds were classified into three groups (>0.13, 0.12 to 0.13, and < 0.12 mm2). Values for each group are expressed as a percentage of the total seed number analyzed.
(B) Seed number per silique for Col-0, da2-1, and 35S:DA2#1. Siliques (from the fourth silique to the tenth silique) on the main stem were used to measure seed number per silique (n = 20).
(C) Seed weight per plant for Col-0, da2-1, and 35S:DA2#1. Seed weight was determined from 15 independent plants for each genotype (n = 15).
(D) Seed number per plant for Col-0, da2-1, and 35S:DA2#1. Seed weight was determined from 15 independent plants for each genotype (n = 15).
(E) Height of Col-0, da2-1, and 35S:DA2#1 plants. Plant height was determined from 20 independent plants for each genotype (n = 20).
(F) Thirty-four-day-old plants of Col-0 (left), da2-1 (middle), and 35S:DA2#1 (right).
(G) Flowers of Col-0 (top), da2-1 (middle), and 35S:DA2#1 (bottom).
Values in (B) to (E) are given as mean ± se relative to the wild-type value, set at 100%. **P < 0.01 and *P < 0.05 compared with the wild type (Student’s t test).
Bars = 1 cm in (F) and 1 mm (G).
[See online article for color version of this figure.]
DA1 and DA2 Act Synergistically to Control Seed Size.
(A) Dry seeds of Col-0, da1-1, da2-1, and da1-1 da2-1.
(B) Ten-day-old seedlings of Col-0, da2-1, da1-1, and da1-1 da2-1 (from left to right).
(C) Seed weight of Col-0, da1-1, da2-1, and da1-1 da2-1. Seed weight was determined by weighing mature dry seeds in batches of 500. The weights of five sample batches were measured for each seed lot (n = 5).
(D) Seed weight of Col-0, da1-ko1, da2-1, and da1-ko1 da2-1. Seed weight was determined by weighing mature dry seeds in batches of 500. The weights of five sample batches were measured for each seed lot (n = 5).
(E) Cotyledon area of 10-d-old Col-0, da1-1, da2-1, and da1-1 da2-1 seedlings (n = 35).
(F) Cotyledon area of 10-d-old Col-0, da1-ko1, da2-1, and da1-ko1 da2-1 seedlings (n = 35).
(G) The average area of palisade cells in cotyledons of Col-0, da1-1, da2-1, and da1-1 da2-1 embryos (n = 120).
(H) Projective area of Col-0, da1-1, da1-1 da2-1, da1-ko1 da2-1, da1-ko1 dar1-1, and da1-ko1 dar1-1 da2-1 seeds (n = 120).
Values in (C) to (H) are given as mean ± se relative to the respective wild-type values, set at 100%. **P < 0.01 and *P < 0.05 compared with the wild type (Student’s t test).
Bars = 0.1 mm in (A) and 1 mm in (B).
[See online article for color version of this figure.]
DA2 Acts Synergistically with DA1 to Regulate Seed Size, but Does So Independently of EOD1
The seeds of the da2-1 mutant were enlarged, but not to the same extent as in da1-1 (Li et al., 2008), suggesting that DA1 and DA2 could function in a common pathway. To test for a genetic interaction between DA1 and DA2, we generated a da1-1 da2-1 double mutant and determined its seed size. Although the da2-1 mutant had slightly larger and heavier seeds than the wild type (Figures 1A, 2C, and 2D; see Supplemental Figure 2B online), the da2-1 mutation synergistically enhanced the seed size and weight phenotypes of da1-1 (Figures 2A and 2C; see Supplemental Figure 2B online), revealing a synergistic genetic interaction between DA1 and DA2 in seed size. The changes in seed size were reflected in the size of the embryos and resulting seedlings (Figure 2B; see Supplemental Figure 2A online). We further measured the cotyledon area of 10-d-old seedlings. A synergistic enhancement of cotyledon size of da1-1 by the da2-1 mutation was also observed (Figures 2B and 2E).
The mutant protein encoded by the da1-1 allele has negative activity toward DA1 and a DA1-related protein (DAR1), its closest homolog (Li et al., 2008). Double da1-knockout1 (da1-ko1) dar1-1 T-DNA insertion mutants exhibited the da1-1 phenotypes, while da1-ko1 and dar1-1 single mutants did not show an obvious seed size phenotype (Li et al., 2008). As da1-1 and da2-1 act synergistically to increase seed size, one would expect that da1-ko1 might synergistically enhance the phenotypes of da2-1. To test this, we generated the da1-ko1 da2-1 double mutant. As shown in Figure 2D and Supplemental Figure 2C online, the seed size and weight phenotypes of da2-1 were also synergistically enhanced by the da1-ko1 mutation. We further measured cotyledon area of 10-d-old seedlings. The da1-ko1 mutation synergistically enhanced the cotyledon size phenotype of da2-1 (Figure 2F). Similarly, a synergistic enhancement of petal size of da2-1 by the da1-ko1 mutation was also observed (see Supplemental Figure 2D online). These results further demonstrate the synergistic effects of the simultaneous disruption of both DA1 and DA2. We further measured the size of embryo cells and petal epidermal cells. Cell size in da1-1 da2-1 and da1-ko1 da2-1 double mutants was not increased, compared with that measured in their parental lines (Figure 2G; see Supplemental Figure 2E online), suggesting that DA1 and DA2 act synergistically to restrict cell proliferation.
The da1-1 da2-1 double mutant had larger seeds than da1-ko1 da2-1 (Figures 2C, 2D, and 2H; see Supplemental Figures 2B and 2C online), which is consistent with our previous report that the da1-1 allele had stronger phenotypes than da1-ko1 (Li et al., 2008). The size of da1-1 seeds was similar to that of da1-ko1 dar1-1 double mutant seeds because the da1-1 allele has a negative effect on DA1 and DAR1 (Figure 2H) (Li et al., 2008). Therefore, one would expect that the size of da1-1 da2-1 double mutant seeds might be similar to that of da1-ko1 dar1-1 da2-1 triple mutant seeds. We therefore generated a da1-ko1 dar1-1 da2-1 triple mutant and investigated its seed size. As shown in Figure 2H, the size of da1-ko1 dar1-1 da2-1 triple mutant seeds was comparable with that of da1-1 da2-1 double mutant seeds, but larger than that of da1-ko1 da2-1 double mutant seeds. Thus, these genetic analyses further support that the da1-1 allele has a negative effect on both DA1 and DAR1 (Li et al., 2008).
We previously identified an enhancer of da1-1 (EOD1), which is allelic to BB (Disch et al., 2006; Li et al., 2008). The eod1 mutations synergistically enhanced the seed size phenotype of da1-1 (Li et al., 2008). Similarly, the seed size and weight phenotypes of da2-1 were synergistically enhanced by da1-1 and da1-ko1 (Figures 2A, 2C, and 2D). We therefore asked whether DA2 and EOD1 could function in a common pathway. To determine the genetic relationship between DA2 and EOD1, we analyzed an eod1-2 da2-1 double mutant. The genetic interaction between eod1-2 and da2-1 was essentially additive for both seed weight and petal size compared with their parental lines (see Supplemental Figures 2F and 2G online), suggesting that DA2 functions to influence seed and organ growth separately from EOD1.
DA2 Acts Maternally to Influence Seed Size
Considering that the size of seeds is affected by the maternal and/or zygotic tissues, we asked whether DA2 functions maternally or zygotically. To test this, we performed reciprocal cross experiments between the wild type and da2-1. As shown in Figure 3E, the effect of da2-1 on seed size was observed only when maternal plants are homozygous for the da2-1 mutation. Seeds produced by maternal da2-1 plants, regardless of the genotype of the pollen donor, were consistently larger than those produced by maternal wild-type plants. This result indicates that da2-1 can act maternally to influence seed size. We previously demonstrated that DA1 also functions maternally to regulate seed size (Li et al., 2008). As the da1-ko1 mutation synergistically enhanced the seed size phenotype of da2-1 (Figure 2D), we further conducted reciprocal cross experiments between the wild type and da1-ko1 da2-1 double mutant. Similarly, the effect of da1-ko1 da2-1 on seed size was observed only when da1-ko1 da2-1 acted as the maternal plant (Figure 3F). Pollinating da1-ko1/+ da2-1/+ plants with da1-ko1 da2-1 double mutant pollen leads to the development of da1-ko1 da2-1, da1-ko1/da1-ko1 da2-1/+, da1-ko1/+ da2-1 da2-1, and da1-ko1/+ da2-1/+ embryos within da1-ko1/+ da2-1/+ seed coats. We further measured the size of individual seeds from da1-ko1/+ da2-1/+ plants fertilized with da1-ko1 da2-1 double mutant pollen and genotyped da1-ko1 and da2-1 mutations. Our results show that da1-ko1 and da2-1 mutations are not associated with variation in the size of these seeds (Figure 3G). Together, these analyses indicate that the embryo and endosperm genotypes for DA1 and DA2 do not affect seed size, and DA1 and DA2 are required in sporophytic tissue of the mother plant to regulate seed growth.
DA1 and DA2 Act Synergistically to Regulate Cell Proliferation in Maternal Integuments of Developing Seeds.
(A) to (D) Mature ovules of Col-0, da1-1, da2-1, and da1-1 da2-1. The da2-1 mutation synergistically enhances the ovule size of da1-1.
(E) Projective area of Col-0 × Col-0 (c/c) F1, da2-1 × da2-1 (d2/d2) F1, Col-0 × da2-1 (c/d2) F1, and da2-1 × Col-0 (d2/c) F1 seeds (n = 120).
(F) Projective area of Col-0 × Col-0 (c/c) F1, da1-ko1 da2-1 × da1-ko1 da2-1 (dd/dd) F1, Col-0 × da1-ko1 da2-1 (c/dd) F1, da1-ko1 da2-1 × Col-0 (dd/c) F1 seeds (n = 120).
(G) Pollinating da1-ko1/+ da2-1/+ plants with da1-ko1 da2-1 double mutant pollen leads to the development of da1-ko1/+ da2-1/+ (a), da1-ko1/+ da2-1da2-1 (b), da1-ko1/da1-ko1 da2-1/+ (c), and da1-ko1 da2-1 (d) embryos within da1-ko1/+ da2-1/+ seed coats. Projective area of individual seeds from da1-ko1/+da2-1/+ plants fertilized with da1-ko1 da2-1 double mutant pollen was measured (n = 50). These seeds were further genotyped for da1-ko1 and da2-1 mutations. The data show that da1-ko1 and da2-1 mutations are not associated with variation in the size of these seeds (P > 0.05, Student’s t test).
(H) Projective area of Col-0, da1-1, da2-1, and da1-1 da2-1 mature ovules (n = 120).
(I) The number of cells in the outer integuments of Col-0, da1-1, da2-1, and da1-1 da2-1 seeds at 6 and 8 DAP. Thirty seeds for each genotype were examined (n = 30).
(J) Average length of cells in the outer integuments of Col-0, da1-1, da2-1, and da1-1 da2-1 seeds at 6 and 8 DAP calculated from the outer integument length and cell number for individual seeds. Thirty seeds for each genotype were examined (n = 30).
Values in (E), (F), and (H) are given as mean ± se relative to the respective wild-type values, set at 100%. **P < 0.01 compared with the wild type (Student’s t test).
Bars = 0.5 mm in (A) to (D).
[See online article for color version of this figure.]
DA2 Acts Synergistically with DA1 to Affect Cell Proliferation in the Maternal Integuments
Reciprocal crosses showed that DA1 and DA2 function maternally to determine seed size (Figures 3E to 3G) (Li et al., 2008). The integuments surrounding the ovule are maternal tissues and form the seed coat, which may physically restrict seed growth, after fertilization. Several studies showed that the integument size of ovules determines seed size (Schruff et al., 2006; Adamski et al., 2009). We therefore asked whether DA1 and DA2 act through the maternal integuments to regulate seed size. To test this, we investigated mature ovules from the wild type, da1-1, da2-1, and da1-1 da2-1 at 2 d after emasculation. The size of da1-1 ovules was dramatically larger than that of wild-type ovules (Figures 3A, 3B, and 3H), consistent with our previous findings (Li et al., 2008). The da2-1 ovules were also larger than wild-type ovules (Figures 3A, 3C, and 3H). The da2-1 mutation synergistically enhanced the ovule size phenotype of da1-1 (Figures 3A to 3D and 3H), consistent with their synergistic interactions in seed size.
The size of the integument or seed coat is determined by cell proliferation and cell expansion. After fertilization, cells in integuments mainly undergo expansion, but still carry on proliferation (Garcia et al., 2005). A previous study showed that the integument of a developing seed ceases cell division at 4 d after pollination (DAP) (Garcia et al., 2005). We therefore investigated the outer integument cell number of developing seeds in the wild type, da1-1, da2-1, and da1-1 da2-1 at 6 and 8 DAP. In wild-type seeds, the number of outer integument cells at 6 DAP was similar to that at 8 DAP (Figure 3I), indicating that cells in the outer integuments of wild-type seeds completely stop division at 6 DAP. Similarly, cells in the outer integuments of da1-1, da2-1, and da1-1 da2-1 seeds completely stopped cell proliferation at 6 DAP (Figure 3I). The number of outer integument cells in da1-1 and da2-1 seeds was significantly increased compared with that in wild-type seeds (Figure 3I). The da2-1 mutation synergistically enhanced the outer integument cell number of da1-1 (Figure 3I). We further investigated the outer integument cell length of wild-type, da1-1, da2-1, and da1-1 da2-1 seeds at 6 and 8 DAP. Cells in da1-1, da2-1, and da1-1 da2-1 outer integuments were significantly shorter than those in wild-type outer integuments (Figure 3J), suggesting a possible compensation mechanism between cell proliferation and cell expansion in the integuments. Thus, these results show that DA2 acts synergistically with DA1 to restrict cell proliferation in the maternal integuments.
DA2 Encodes a Functional E3 Ubiquitin Ligase
The da2-1 mutation was identified as having a T-DNA insertion in the seventh exon of the gene At1g78420 (Figure 4A). The T-DNA insertion site was further confirmed by PCR using T-DNA–specific and flanking primers and sequencing the PCR products (see Supplemental Figures 3A and 3B online). The full-length mRNA of At1g78420 could not be detected in da2-1 mutant (see Supplemental Figure 3C online). We expressed At1g78420 coding DNA sequence under the control of its own promoter in da2-1 plants and isolated 62 transgenic plants. Nearly all transgenic lines exhibited complementation of da2-1 phenotypes (Figures 4D to 4F), indicating that At1g78420 is DA2.
Identification and Molecular Characterization of DA2.
(A) The DA2 gene structure. The start codon (ATG) and the stop codon (TAA) are indicated. Closed boxes indicate the coding sequence, open boxes indicate the 5′ and 3′ untranslated regions, and lines between boxes indicate introns. The T-DNA insertion site (da2-1) in DA2 is shown.
(B) DA2 contains a predicted RING domain. aa, amino acids.
(C) E3 ubiquitin ligase activity of DA2. MBP-DA2 and mutated DA2 (MBP-DA2C59S and MBP-DA2N91L) fusion proteins were assayed for E3 ubiquitin ligase activity in the presence of E1, E2, and His-ubiquitin (His-Ub). Ubiquitinated proteins were detected by immunoblotting (IB) with anti-His antibody and anti-MBP antibody, respectively. The black arrow indicates MBP-DA2 proteins, and the red arrow shows ubiquitinated MBP-DA2 proteins.
(D) Projective area of Col-0, da2-1, COM#6, COM#8, and COM#10 seeds (n = 120). COM is da2-1 transformed with the DA2 coding sequence driven by its own promoter.
(E) Petal area of Col-0, da2-1, COM#6, COM#8, and COM#10 plants (n = 50).
(F) Quantitative real-time RT-PCR analysis of DA2 expression in Col-0, da2-1, COM#6, COM#8, and COM#10 seedlings. Three biological replicates, each consisting of three technical replicates, were performed (n = 3).
Values in (D) to (F) are given as mean ± se relative to the da2-1 values, set at 100%. **P < 0.01 compared with the da2-1 mutant (Student’s t test).
[See online article for color version of this figure.]
To further characterize DA2 function, in particular gain-of-function phenotypes, we expressed the coding region of DA2 under the control of the cauliflower mosaic virus 35S promoter in wild-type plants and isolated 77 transgenic plants. Overexpression of DA2 caused decreases in seed size, seed yield per plant, and seed number per plant (Figures 1A, 1C, and 1D). In addition, most transgenic plants overexpressing DA2 had small flowers and leaves, short siliques, reduced plant height, as well as decreased biomass compared with the wild type (Figures 1E to 1G; see Supplemental Figures 1A to 1C and Supplemental Figure 4 online). These results further support the role of DA2 in limiting seed and organ growth.
DA2 is predicted to encode a 402–amino acid protein containing one predicted RING domain (59-101) (Figure 4B; see Supplemental Figure 5 online). Previous research showed that RING-type proteins can act as E3 ubiquitin ligases that function as adapters that transfer ubiquitin from E2 ubiquitin ligases to target proteins (Seo et al., 2003). To investigate whether DA2 has E3 ubiquitin ligase activity, we expressed DA2 in Escherichia coli as a fusion protein with maltose binding protein (MBP) and purified MBP-DA2 protein from the soluble fraction. In the presence of an E1 ubiquitin activating enzyme, an E2-conjugating enzyme, His-ubiquitin, and MBP-DA2, a polyubiquitination signal, were observed by immunoblot using an anti-His antibody (Figure 4C, fifth lane from the left). The anti-MBP blot analysis also showed that MBP-DA2 was ubiquitinated (Figure 4C, fifth lane from the left). However, in the absence of E1, E2, His-ubiquitin, or MBP-DA2, no polyubiquitination was detected (Figure 4C, first to fourth lanes from the left), demonstrating that DA2 is a functional E3 ubiquitin ligase.
The RING motif is essential for the E3 ubiquitin ligase activity of RING finger proteins (Xie et al., 2002). Therefore, we tested whether an intact RING finger domain was required for DA2 E3 ligase activity. A single amino acid substitution allele was produced by mutagenizing Cys-59 to Ser (C59S), as this mutation is predicted to disrupt the RING domain (see Supplemental Figure 5 and Supplemental Data Set 1 online). An in vitro ubiquitination assay indicated that the E3 ligase activity was abolished in the C59S mutant of DA2 (Figure 4C, sixth lane from the left), indicating that an intact RING domain is required for DA2 E3 ubiquitin ligase activity. We further overexpressed DA2C59S (35S:DA2C59S) in wild-type Columbia-0 (Col-0) plants and isolated 69 transgenic plants. The seed and petal size of transgenic plants was comparable with that of wild-type plants, although transgenic plants had high expression levels of DA2C59S (see Supplemental Figures 6A to 6C online), indicating that the DA2C59S mutation affects the function of DA2 in seed and organ growth.
Three RING types, RING-H2, RING-HCa, and RING-HCb, and five modified RING types, RING-C2, RING-v, RING-D, RING-S/T, and RING-G, have been described in Arabidopsis (Stone et al., 2005). A new type of RING domain (C5HC2) found in rice GW2 has been proposed (Song et al., 2007). Although the spacing of the Cys residues in the predicted RING domain of DA2 was similar to that in the RING domain (C5HC2) of rice GW2, the RING domain of DA2 lacked a conserved His residue that was replaced by an Asn residue (Asn-91) (see Supplemental Figure 5 and Supplemental Data Set 1 online). This amino acid substitution was also observed in the predicted RING domain of DA2 homologs in dicots, such as soybean (Glycine max) and oilseed rape (Brassica napus) (see Supplemental Figure 5 and Supplemental Data Set 1 online). We therefore asked whether this Asn residue (Asn-91) is crucial for its E3 ubiquitin ligase activity. A single amino acid substitution allele was produced by mutagenizing Asn-91 to Leu (N91L). An in vitro ubiquitination assay showed that the N91L mutant of DA2 had the E3 ligase activity (Figure 4C, the seventh lane from the left), suggesting that Asn-91 may not be required for DA2 E3 ligase activity. These results suggest that the RING domain of DA2 might be a variant of that found in GW2. We further overexpressed DA2N91L (35S:DA2N91L) in wild-type plants and isolated 26 transgenic plants. The seeds of transgenic plants were smaller than wild-type seeds (see Supplemental Figures 6D and 6E online), suggesting that the DA2N91L could restrict seed growth.
Homologs of Arabidopsis DA2
Proteins that share significant homology with DA2 outside of the RING domain are found in Arabidopsis and crop plants, including oilseed rape, soybean, rice, maize (Zea mays), and barley (Hordeum vulgare) (see Supplemental Data Set 1 and Supplemental Figure 7 online). One predicted protein in Arabidopsis shares extensive amino acid similarity with DA2 and is named DA2-like protein (DA2L; At1g17145) (see Supplemental Data Set 1 and Supplemental Figure 7 online). Like 35S:DA2 plants, DA2L-overexpressing lines exhibited small plants and organs (see Supplemental Figure 8 online), suggesting that DA2 and DA2L have similar functions. The similar proteins in other plant species show a 39.2 to 84.5% amino acid sequence identity with DA2 (see Supplemental Data Set 1 and Supplemental Figure 7 online). The homolog in oilseed rape had the highest amino acid sequence identity with DA2 (84.5%) (see Supplemental Data Set 1 and Supplemental Figure 7 online). Rice GW2 had 43.1% amino acid sequence identity with Arabidopsis DA2 (see Supplemental Data Set 1 and Supplemental Figure 7 online). As overexpression of GW2 reduced grain width in rice (Song et al., 2007), we asked whether DA2 and GW2 perform similar functions in seed size regulation. We therefore overexpressed GW2 in wild-type plants. Like 35S:DA2 and 35S:DA2L transgenic lines, the Arabidopsis transgenic plants overexpressing GW2 produced smaller seeds and organs than wild-type plants (see Supplemental Figure 9 online), suggesting a possible conserved function for Arabidopsis DA2 and rice GW2 in seed and organ growth regulation.
DA2 and DA1 Show Similar Expression Patterns
To determine the expression pattern of DA2, RNA from roots, stems, leaves, seedlings, and inflorescences was analyzed by quantitative real-time RT-PCR analysis. DA2 mRNA was detected in all plant organs tested (Figure 5A). The tissue-specific expression patterns of DA2 were investigated using histochemical assay of β-glucuronidase (GUS) activity of transgenic plants containing a DA2 promoter:GUS fusion (pDA2:GUS). GUS activity was detected in roots, cotyledons, leaves, and inflorescences (Figures 5B and 5C). Relatively high GUS activity was detected in leaf primordia and roots (Figures 5B and 5C). In flowers, relatively stronger expression of DA2 was observed in young floral organs than old floral organs (Figures 5D to 5L). Similarly, higher GUS activity was detected in younger ovules than older ones (Figures 5M and 5N). This shows that DA2 expression is regulated temporally and spatially. We previously reported that DA1 was highly expressed during early stages of organ formation, but levels were largely reduced at the later stages of organ development (Li et al., 2008). Together, these results suggest that DA1 and DA2 have similar expression patterns during organ development.
Expression Patterns of DA2.
(A) Quantitative real-time RT-PCR analysis of DA2 expression. Total RNA was isolated from roots (R), stems (S), leaves (L), seedlings (Se), and inflorescences (In).
(B) to (N) DA2 expression activity was monitored by pDA2:GUS transgene expression. Four GUS-expressing lines were observed, and all showed a similar pattern, although they differed slightly in the intensity of the staining. Histochemical analysis of GUS activity in a 4-d-old seedling (B), a 10-d-old seedling (C), a floral inflorescence (D), the developing petals ([E] to [G]), the developing stamens ([H] and [I]), the developing carpels ([J] to [L]), and the developing ovules ([M] and [N]). Bars =1 mm in (B) to (D) and 0.1 mm in (E) to (N).
[See online article for color version of this figure.]
DA1 Interacts with DA2 in Vitro and in Vivo
Our genetic analyses show that DA1 acts synergistically with DA2 to restrict seed and organ growth. Such synergies can be caused by simultaneously disrupting two components of a protein complex (Pérez-Pérez et al., 2009; Lanctot et al., 2013). In addition, ubiquitin receptors have been reported to interact with E3 ubiquitin ligases and influence the degradation of their ubiquitinated substrates in animals (Alexandru et al., 2008; Bandau et al., 2012). We therefore assessed whether DA1 interacts with the E3 ubiquitin ligase DA2 using an in vitro interaction/pull-down experiment. DA1 was expressed as a glutathione S-transferase (GST) fusion protein, while DA2 was expressed as a MBP fusion protein. As shown in Figure 6A (first and second lanes from left), GST-DA1 bound to MBP-DA2, while GST-DA1 did not bind to a negative control (MBP). This result indicates that DA1 physically interacts with DA2 in vitro.
DA1 Interacts with DA2 in Vitro and in Vivo.
(A) DA1 directly interacts with DA2 in vitro. GST-DA1, GST-DA1R358K, GST-DA1-UIM, GST-DA1-LIM, GST-DA1-LIM+C, and GST-DA1-C were pulled down (PD) by MBP-DA2 immobilized on amylose resin and analyzed by immunoblotting (IB) using an anti-GST antibody.
(B) Schematic diagram of DA1 and its derivatives containing specific protein domains. The predicted DA1 protein contains two UIM motifs, a single LIM domain, and the C-terminal region. aa, amino acids.
(C) DA1 interacts with DA2 in vivo. N. benthamiana leaves were transformed by injection of Agrobacterium GV3101 cells harboring 35S:Myc-DA1 and 35S:GFP-DA2 plasmids. Total proteins were immunoprecipitated with GFP-Trap-A, and the immunoblot was probed with anti-GFP and anti-Myc antibodies, respectively.
Myc-DA1 was detected in the immunoprecipitated GFP-DA2 complex, indicating that there is a physical association between DA1 and DA2 in planta.
[See online article for color version of this figure.]
DA1 contains two UIMs, a single LIM domain defined by its conservation with the canonical Lin-11, Isl-1, and Mec3 domains and the highly conserved C-terminal region (Figure 6B) (Li et al., 2008). We further asked which domain of DA1 is required for the interaction between DA1 and DA2. A series of DA1 derivatives containing specific protein domains were expressed in E. coli: DA1-UIM, containing only the two UIM domains; DA1-LIM, with only the LIM domain; DA1-LIM+C, containing only the LIM domain and the C-terminal region; and DA1-C, with only the C-terminal region, were expressed as GST fusion proteins (Figure 6B). DA2 was expressed as an MBP fusion protein and used in pull-down experiments. As shown in Figure 6A, GST-DA1-LIM+C and GST-DA1-C interacted with MBP-DA2, but GST-DA1-UIM and GST-DA1-LIM did not bind to MBP-DA2. This result indicates that the conserved C-terminal region of DA1 interacts with DA2.
Considering that the mutant protein encoded by the da1-1 allele (DA1R358K) has a mutation in the C-terminal region (Figure 6B) (Li et al., 2008), we asked whether the DA1R358K mutation affects interactions with DA2. Using a GST-DA1R358K fusion protein in pull-down experiments with MBP-DA2, we showed that the mutation in DA1R358K does not affect the interaction between DA1 and DA2 (Figure 6A, third lane from left).
To further investigate the possible association between DA1 and DA2 in planta, we used coimmunoprecipitation analysis to detect their interactions in vivo. We transiently coexpressed 35S:Myc-DA1 and 35S:GFP-DA2 in Nicotiana benthamiana leaves. Transient coexpression of 35S:GFP and 35S:Myc-DA1 in N. benthamiana leaves was used as a negative control. Total proteins were isolated and incubated with GFP-Trap-A agarose beads to immunoprecipitate GFP-DA2 or GFP. Precipitates were detected with anti-GFP and anti-Myc antibodies, respectively. As shown in Figure 6C, Myc-DA1 was detected in the immunoprecipitated GFP-DA2 complex but not in the negative control (GFP), indicating that there is a physical association between DA1 and DA2 in planta. As the C-terminal region of DA1 interacted with DA2 in the pull-down assay (Figure 6A), we further asked whether the C terminus of DA1 interacts with DA2 in planta. The coimmunoprecipitation analysis showed that the C-terminal region of DA1 (Myc-DA1-C) was detected in the GFP-DA2 complex but not in the negative control (PEX10-GFP, a RING-type E3 ubiquitin ligase) (see Supplemental Figure 10 online) (Platta et al., 2009; Kaur et al., 2013). Thus, these results indicate that the C-terminal region of DA1 is required for the interaction with DA2 in vitro and in vivo.
DISCUSSION
Seed size in higher plants is a key determinant of evolutionary fitness and is an important agronomic trait in crop domestication (Gómez, 2004; Orsi and Tanksley, 2009). Several factors that act maternally to regulate seed size have been identified, such as ARF2/MNT, AP2, KLU/CYP78A5, EOD3/CYP78A6, and DA1. However, the genetic and molecular mechanisms of these factors in seed size regulate are nearly totally unknown. We previously demonstrated that the ubiquitin receptor DA1 acts synergistically with the E3 ubiquitin ligase EOD1/BB to regulate seed size (Li et al., 2008). In this study, we identified Arabidopsis DA2 as another RING E3 ubiquitin ligase involved in regulating seed size. Genetic analyses show that DA2 functions synergistically with DA1 to regulate final seed size, but does so independently of the E3 ubiquitin ligase EOD1. We further revealed that DA1 interacts physically with DA2. Our results define a ubiquitin-based system involving DA1, DA2, and EOD1 that controls final seed size in Arabidopsis.
DA2 Acts Maternally to Regulate Seed Size
The da2-1 loss-of-function mutant formed large seeds and organs, whereas plants overexpressing DA2 produced small seeds and organs (Figures 1A, 1F, and 1G), indicating that DA2 is a negative factor of seed and organ size regulation. Surprisingly, Arabidopsis DA2 was recently proposed to function as a positive regulator of organ growth, although nothing is known about how DA2 regulates seed and organ growth (Van Daele et al., 2012). In this study, we have sufficient evidence to prove that DA2 is a negative regulator of seed and organ growth. The da2-1 loss-of-function mutant formed large seeds and organs (Figures 1 and 2). Supporting this, the da2-1 mutation synergistically enhanced the seed and organ size phenotypes of da1-1 and da1-ko1 (Figures 1 and 2; see Supplemental Figures 2A to 2D online). The da2-1 mutation also enhanced the seed and organ size phenotypes of eod1-2 (see Supplemental Figures 2F and 2G online), further indicating that the da2-1 mutation promotes seed and organ growth. The da2-1 mutant formed large ovules with more cells in the integuments, and the da2-1 mutation synergistically enhanced the ovule size phenotype of da1-1 (Figures 3H and 3I). In addition, most transgenic plants overexpressing DA2 and DA2L were smaller than wild-type plants (Figures 1F and 1G; see Supplemental Figure 9 online). The organ growth phenotypes of these transgenic plants were correlated with their respective expression levels (see Supplemental Figures 4 and 9 online). Therefore, our data demonstrate that DA2 functions as a negative regulator of seed and organ size. Several Arabidopsis mutants with large organs also formed large seeds (Krizek, 1999; Mizukami and Fischer, 2000; Schruff et al., 2006; Li et al., 2008; Adamski et al., 2009), suggesting a possible link between organ size and seed growth. By contrast, several other mutants with large organs exhibited normal sized seeds (Hu et al., 2003; White, 2006; Xu and Li, 2011), indicating that organ and seed size is not invariably positively related. These results suggest that seeds and organs have both common and distinct pathways that regulate their respective size.
Reciprocal cross experiments showed that DA2 acts maternally to influence seed growth, and the embryo and endosperm genotypes for DA2 do not affect seed size (Figures 3E to 3G). The integuments surrounding the ovule are maternal tissues and form the seed coat after fertilization. Alterations in maternal integument size, such as those seen in arf2, da1-1, and klu ovules, have been known to contribute to changes in seed size (Schruff et al., 2006; Li et al., 2008; Adamski et al., 2009). Mature da2-1 ovules were larger than mature wild-type ovules (Figures 3A, 3C, and 3H). The da2-1 mutation also synergistically enhanced the integument size of da1-1 ovules (Figures 3A to 3D and 3H). Thus, a general theme emerging from these studies is that the regulation of maternal integument size is a key mechanism in determining final seed size. Consistent with this notion, plant maternal factors of seed size regulation (e.g., KLU, ARF2, and DA1) isolated to date influence integument size (Schruff et al., 2006; Li et al., 2008; Adamski et al., 2009).
The size of the integument or seed coat is determined by cell proliferation and cell expansion, two processes that are coordinated. Cell number in the integuments of the mature ovule sets the growth potential of the seed coat after fertilization. For example, arf2 mutants produced large ovules with more cells, leading to large seeds (Schruff et al., 2006), while klu mutants had small ovules with fewer cells, resulting in small seeds (Adamski et al., 2009). Our results show that the integuments of da1-1 and da2-1 seeds had more cells than those of wild-type seeds, and da1-1 and da2-1 act synergistically to promote cell proliferation in the integuments (Figure 3I). We also observed that cells in the outer integuments of da1-1, da2-1, and da1-1 da2-1 seeds were shorter than those in wild-type integuments (Figure 3J), suggesting the existence of a possible compensation mechanism between cell proliferation and cell elongation in the maternal integument. Thus, it is possible that the maternal integument or seed coat, which acts as a physical constraint on seed growth, can set an upper limit to final seed size.
A Genetic Framework for Ubiquitin-Mediated Regulation of Seed Size
DA2 encodes a protein with one predicted RING domain that is distinctive from any of the previously described plant RING domains. The RING domain of DA2 shared the highest homology (81.4% identity) with that of rice GW2 (C5HC2), but it lacked one conserved metal ligand amino acid (a His residue) that was replaced by an Asn residue (see Supplemental Figure 4 and Supplemental Data Set 1 online) (Song et al., 2007). It is still possible that the RING domain of DA2 might be a variant of that found in GW2. Many RING-type domains are found in E3 ubiquitin ligases that ubiquitinate substrates, often targeting them for subsequent proteasomal degradation (Smalle and Vierstra, 2004). We tested the E3 activity of recombinant DA2 in an in vitro ubiquitin-ligase assay and demonstrated that DA2 is a functional E3 ubiquitin ligase (Figure 4C), suggesting that DA2 may target positive regulators of cell proliferation for ubiquitin-dependent degradation by the 26S proteasome. Proteins that share homology with DA2 outside the RING domain are found in Arabidopsis and other plant species (see Supplemental Data Set 1 and Supplemental Figure 7 online). In Arabidopsis, the DA2L protein shares extensive amino acid similarity with DA2 (see Supplemental Data Set 1 online). Like 35S:DA2 plants, DA2L-overexpressing lines showed small plants (see Supplemental Figure 8 online), suggesting that DA2 and DA2L may perform similar functions. The homolog of DA2 in rice is the RING-type (C5HC2) protein GW2 (Song et al., 2007), which has been known to act as a negative regulator of seed size. However, the genetic and molecular mechanisms of GW2 in seed size regulation are largely unknown in rice.
We previously identified DA1, a ubiquitin receptor with ubiquitin binding activity, as a negative regulator of seed size (Li et al., 2008). A modifier screen identified EOD1 (Li et al., 2008), which is allelic to the E3 ubiquitin ligase BB (Disch et al., 2006). Analysis of double eod1-2 da1-1 mutants revealed synergistic genetic interactions between DA1 and EOD1 (Li et al., 2008), suggesting that these genes may control regulate seed growth by modulating the activity of a common target(s). Although genetic interactions between da1-1 and eod1-2 also synergistically enhanced seed and organ size, our genetic analyses show that DA2 acts independently of EOD1 to influence seed growth, suggesting that DA2 and EOD1 may target distinct growth stimulators for degradation, with common regulation via DA1. Thus, our findings establish a framework for the regulation of seed and organ size by three ubiquitin-related proteins: DA1, DA2, and EOD1. In addition, we observed that overexpression of GW2 restricts seed and organ growth in Arabidopsis, suggesting a possible conserved function in Arabidopsis and rice. It would be interesting to investigate the effects of the combination of GW2 and rice homologs of DA1 and EOD1 on grain size in rice.
A Possible Molecular Mechanism of DA1 and DA2 in Seed Size Regulation
Our results demonstrate that the E3 ubiquitin ligase DA2 interacts with the ubiquitin receptor DA1 in vitro and in vivo (Figure 6). However, it is not likely that DA2 targets DA1 for proteasomal degradation because a T-DNA inserted mutant of DA1 (da1-ko1) synergistically enhances the seed size phenotype of da2-1 (Figures 2D and 2F; see Supplemental Figures 2C and 2D online). Nevertheless, many other types of ubiquitin modification regulate proteins in a proteasome-independent manner (Schnell and Hicke, 2003). For example, monoubiquitination has been implicated in the activation of signaling proteins, endocytosis, and histone modification (Schnell and Hicke, 2003). In animals, monoubiquitination of the ubiquitin receptor eps15 depends on the interaction between eps15 and the Nedd4 family of E3 ligases (Woelk et al., 2006). By contrast, an E3-independent monoubiquitination of ubiquitin receptors has also been reported (Hoeller et al., 2007). Considering that DA1 interacts with DA2, we tested whether DA2 can ubiquitinate or monoubiquitinate DA1. In the presence of E1, E2, and ubiquitin, DA2-His had an E3 ubiquitin ligase activity (see Supplemental Figure 11A online, fifth lane from the left). However, in the presence of E1, E2, ubiquitin, and DA2-His (E3), no ubiquitinated DA1-HA was detected under our reaction conditions (see Supplemental Figure 11B online, sixth lane from the left), suggesting that DA2 might not ubiquitinate DA1.
Ubiquitin receptors can interact with polyubiquitinated substrates of E3s via UIM domains and facilitate their degradation by the proteasome (Verma et al., 2004). We previously demonstrated that UIM domains of DA1 can bind ubiquitin (Li et al., 2008). Taken together with its interaction with DA2 through its C-terminal region (Figures 6A and 6B; see Supplemental Figure 10 online), it is possible that DA1 is involved in mediating the degradation of the ubiquitinated substrates of DA2 by the proteasome. One mechanism may involve an interaction between DA1 and DA2, which helps DA1 specifically recognize the ubiquitinated substrate(s) of DA2. DA1 may subsequently bind the polyubiquitin chains of the ubiquitinated substrate(s) through its UIM domain and mediate the degradation of the ubiquitinated substrate(s).
Improving seed yield is an important target for crop breeders worldwide, and the size of seeds is an important component of overall seed yield. We identified DA2 as an important regulator of seed size that functions synergistically with DA1 to influence seed size. DA1 also acts synergistically with EOD1 to affect seed growth. Overexpression of a dominant-negative da1-1 mutation (Zea mays da1-1) has been reported to increase seed mass of maize (Wang et al., 2012), indicating the promise of combining the effects of DA1, DA2, and EOD1 from different seed crops to engineer large seed size in these crops.
METHODS
Plant Materials and Growth Conditions
Arabidopsis thaliana ecotype Columbia (Col-0) was the wild-type line used. All mutants were in the Col-0 background. da2-1 (SALK_150003) was obtained from the Nottingham Arabidopsis Stock Centre and ABRC collections. The T-DNA insertion was confirmed by PCR and sequencing using the primers described in Supplemental Table 1 online. Seeds were surface-sterilized with 100% isopropanol for 1 min and 10% (v/v) household bleach for 10 min, washed at least three times with sterile water, stratified at 4°C for 3 d in the dark, dispersed on GM medium with 0.9% agar and 1% Glc, and then grown at 22°C. Plants were grown under long-day conditions (16-h light/8-h dark) at 22°C.
Constructs and Transformation
The pDA2:DA2 construct was made using a PCR-based Gateway system. The 1960-bp promoter sequence of DA2 was amplified using the primers DA2proGW-F and DA2proGW-R. PCR products were then cloned into the pCR8/GW/TOPO TA cloning vector (Invitrogen). The DA2 coding DNA sequence was amplified using the primers DA2CDSAK-F and DA2CDSAK-R (see Supplemental Table 1 online). PCR products were then cloned into the AscI and KpnI sites of the Gateway vector pMDC110 to yield the DA2CDS-pMDC110 plasmid. The DA2 promoter was then subcloned into DA2CDS-pMDC110 by the attL × attR reaction to generate the pDA2:DA2 construct. The plasmid pDA2:DA2 was introduced into da2-1 mutant plants using Agrobacterium tumefaciens GV3101, and transformants were selected on medium containing hygromycin (30 µg/mL).
The 35S:DA2 construct was made using a PCR-based Gateway system. The specific primers used for the 35S:DA2 construct are DA2CDS-F and DA2CDS-R (see Supplemental Table 1 online). PCR products were subcloned into the pCR8/GW/TOPO TA cloning vector (Invitrogen) using TOPO enzyme. DA2 was then subcloned into Gateway binary vector pMDC32 containing the 35S promoter (Curtis and Grossniklaus, 2003). The plasmid 35S:DA2 was introduced into Col-0 plants using Agrobacterium GV3101, and transformants were selected on medium containing hygromycin (30 µg/mL).
The 1960-bp promoter sequence of DA2 was amplified using the primers DA2proSN-F and DA2proSN-R (see Supplemental Table 1 online). PCR products were cloned into the pGEM-T vector (Promega) using T4 DNA ligase and sequenced. The DA2 promoter was then inserted into the SacI and NcoI sites of the binary vector pGreen-GUS (Curtis and Grossniklaus, 2003) to generate the transformation plasmid pDA2:GUS. The plasmid pDA2:GUS was introduced into Col-0 plants using Agrobacterium GV3101, and transformants were selected on medium containing kanamycin (50 µg/mL).
The 35S:GW2 construct was made using a PCR-based Gateway system. The specific primers used for the 35S:GW2 construct are GW2CDS-F and GW2CDS-R (see Supplemental Table 1 online). PCR products were subcloned into the pCR8/GW/TOPO TA cloning vector (Invitrogen) using TOPO enzyme. GW2 was then subcloned into Gateway binary vector pMDC32 containing the 35S promoter (Curtis and Grossniklaus, 2003). The plasmid 35S:GW2 was introduced into Col-0 plants using Agrobacterium GV3101, and transformants were selected on medium containing hygromycin (30 µg/mL).
Morphological and Cellular Analysis
Average seed weight was determined by weighing mature dry seeds in batches of 500 using an electronic analytical balance (Mettler Moledo AL104). The weights of five sample batches were measured for each seed lot. Seeds were photographed under a Leica microscope (S8APO) and a Leica charge-coupled device (DFC420), and seed size was measured using Image J software.
Area measurements of petals (stage 14), leaves, and cotyledons were made by scanning organs to produce a digital image and then calculating area, length, and width using Image J software. Leaf, petal, and embryo cell sizes were measured from differential interference contrast images. Biomass accumulation in flowers (stage 14) was measured by weighing organs.
GUS Staining
Samples (pDA2:GUS) were stained in a solution of 1 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid, 100 mM Na3PO4 buffer, 3 mM each K3Fe(CN)6/K4Fe(CN)6, 10 mM EDTA, and 0.1% Nonidet P-40 and incubated at room temperature for 6 h. After GUS staining, chlorophyll was removed using 70% ethanol.
RNA Isolation, RT-PCR, and Quantitative Real-Time RT-PCR Analysis
Total RNA was extracted from Arabidopsis roots, stems, leaves, seedlings, and inflorescences using an RNeasy plant mini kit (Tiangen). RT-PCR was performed as described (Li et al., 2006). cDNA samples were standardized based on the amount of ACTIN2 transcript using the primers ACTIN2-F and ACTIN2-R (see Supplemental Table 1 online). Quantitative real-time RT-PCR analysis was performed with a LightCycler 480 engine (Roche) using the LightCycler 480 SYBR Green Master (Roche). ACTIN7 mRNA was used as an internal control, and relative amounts of mRNA were calculated using the comparative threshold cycle method. The primers used for RT-PCR and real-time RT-PCR are described in Supplemental Table 1 online.
E3 Ubiquitin Ligase Activity Assay
The coding sequence of DA2 was cloned into the BamHI and PstI sites of the pMAL-C2 vector to generate the construct MBP-DA2. The specific primers for MBP-DA2 were MBP-DA2-F and MBP-DA2-R (see Supplemental Table 1 online). Mutated DA2 (DA2C59S and DA2N91L) was generated by following the instruction manual of a multi-site-directed mutagenesis kit (Stratagene).
Bacterial lysates expressing MBP-DA2 and mutated MBP-DA2 were prepared from Escherichia coli BL21 induced with 0.4 mM isopropyl β-d-1-thiogalactopyranoside for 2 h. Bacteria were lysed in Triton Glycerol HEPES lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X-100, 10% glycerol, and protease inhibitor cocktail [Roche]) and sonicated. The lysates were cleared by centrifugation and incubated with amylose resin (New England Biolabs) at 4°C for 30 min. Beads were washed by column buffer (20 mM Tris, pH 7.4, 200 mM NaCl, and 1 mM EDTA) and equilibrated by reaction buffer (50 mM Tris, pH 7.4, 20 mM DTT, 5 mM MgCl2, and 2 mM ATP). Then, 110 ng E1 (Boston Biochem), 170 ng E2 (Boston Biochem), 1 μg His-ubiquitin (Sigma-Aldrich), and 2 μg DA2-MBP or mutated DA2-MBP fusion protein were incubated in a 20-μL reaction buffer for 2 h at 30°C. Polyubiquitinated proteins were detected by immunoblotting with an antibody against His (Abmart) and an antibody against MBP (New England Biolabs).
In Vitro Protein–Protein Interaction
The coding sequences of DA1, da1-1, and DA1 derivatives containing specific protein domains were cloned into BamHI and NotI sites of the pGEX-4T-1 vector to generate GST-DA1, GST-DA1R358K, GST-DA1-UIM, and GST-DA1-LIM+C constructs, and EcoRI and XhoI sites of the pGEX-4T-1 vector to generate GST-DA1-LIM and GST-DA1-C constructs. The specific primers for GST-DA1 and GST-DA1R358K were GST-DA1-F and GST-DA1-R (see Supplemental Table 1 online). The specific primers for GST-DA1-UIM, GST-DA1-LIM, GST-DA1-LIM+C, and GST-DA1-C were UIM-F, UIM-R, LIM-F, LIM-R, LIM+C-F, LIM+C-R, C-F, and C-R, respectively (see Supplemental Table 1 online).
To test protein–protein interaction, bacterial lysates containing ∼15 μg of MBP-DA2 fusion proteins were combined with lysates containing ∼30 μg of GST-DA1, GST-DA1R358K, GST-DA1-UIM, GST-DA1-LIM, GST-DA1-LIM+C, or GST-DA1-C fusion proteins. Twenty microliters of amylose resin (New England Biolabs) was added into each combined solution with continued rocking at 4°C for 1 h. Beads were washed five times with TGH buffer, and the isolated proteins were separated on a 10% SDS-polyacrylamide gel and detected by immunoblot analysis with anti-GST (Abmart) and anti-MBP antibodies (Abmart), respectively.
Coimmunoprecipitation
The coding sequence of DA1 and DA1-C was cloned into the KpnI and BamHI sites of the pCAMBIA1300-221-Myc vector to generate the transformation plasmid 35S:Myc-DA1 and 35S:Myc-DA1-C. The specific primers used to amplify Myc-DA1 and Myc-DA1-C were Myc-DA1-F, Myc-DA1-R, Myc-DA1-C-F, and Myc-DA1-C-R, respectively (see Supplemental Table 1 online). The specific primers used to amplify 35S:GFP-DA2 were DA2CDS-F and DA2CDS-R (see Supplemental Table 1 online). PCR products were subcloned into the pCR8/GW/TOPO TA cloning vector (Invitrogen) using TOPO enzyme. DA2 was then subcloned into Gateway binary vector pMDC43 containing the 35S promoter and GFP (Curtis and Grossniklaus, 2003). The specific primers used for the 35S:PEX10-GFP construct are PEX10-F and PEX10-R (see Supplemental Table 1 online). PCR products were subcloned into the pCR8/GW/TOPO TA cloning vector (Invitrogen) using TOPO enzyme. PEX10 was then subcloned into Gateway binary vector pH7FWG2 containing the 35S promoter and GFP.
Nicotiana benthamiana leaves were transformed by injection of Agrobacterium GV3101 cells harboring 35S:Myc-DA1 and 35S:GFP-DA2 plasmids as previously described (Voinnet et al., 2003). Total protein was extracted with extraction buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 20% glycerol, 2% Triton X-100, 1 mM EDTA, 1× Complete protease inhibitor cocktail [Roche], and 20 μg/mL MG132) and incubated with GFP-Trap-A (Chromotek) for 1 h at 4°C. Beads were washed three times with wash buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, and 1× Complete protease inhibitor cocktail [Roche]). The immunoprecipitates were separated in 10% SDS-polyacrylamide gel and detected by immunoblot analysis with anti-GFP (Beyotime) and anti-Myc (Abmart) antibodies, respectively.
Accession Numbers
Arabidopsis Genome Initiative locus identifiers for genes mentioned in this article are as follows: At1g19270 (DA1), At4g36860 (DAR1), At1g78420 (DA2), At1g17145 (DA2L), and At3g63530 (EOD1/BB). Other sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: Os_GW2 (ABO31101.1), Bd_Bradi3g09270 (XP_003571977), Hv_Yrg1 (ABY51682), Zm_gi|220961719 (ACL93316), Sb_gi|242064618 (XP_002453598), At_DA2 (NP_565180), At_DA2L (NP_564016), Rc_gi|255578534 (XP_002530130), Pt_gi|224061326 (XP_002300427), Vv_DA2 (CAN66658), Gm_Glyma13g33260.1 (XP_003543150), Sb_Sb10g003820 (XP_002436503), Zm_gi|260935347 (ACX54355), Os_gi|218197613 (EEC80040), and Bd_Bradi1g49080 (XP_003564311).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Organ Size in the da2-1 Mutant.
Supplemental Figure 2. DA1 and DA2 Act Synergistically to Regulate Seed Size.
Supplemental Figure 3. Molecular Characterization of the DA2 Gene.
Supplemental Figure 4. Overexpression of DA2 Restricts Organ Growth.
Supplemental Figure 5. Alignment of RING Domain of DA2 Homologs.
Supplemental Figure 6. The Seed Size Phenotype of 35S:DA2C59S and 35S:DA2N91L.
Supplemental Figure 7. Phylogenetic Tree of DA2 Homologs.
Supplemental Figure 8. Overexpression of DA2L Restricts Organ Growth.
Supplemental Figure 9. Overexpression of GW2 Restricts Seed and Organ Growth.
Supplemental Figure 10. The C-Terminal Region of DA1 Interacts with DA2 in Planta.
Supplemental Figure 11. DA2 Has E3 Ubiquitin Ligase Activity, but May Not Ubiquitinate DA1.
Supplemental Table 1. Primers Used in This Study.
Supplemental Data Set 1. Alignment of DA2 Homologs Used to Generate Phylogeny Presented in Supplemental Figure 7 Online.
Acknowledgments
We thank the anonymous reviewers and the editor for their critical comments on this article, Qi Xie for his suggestions on ubiquitination assay, and the ABRC and the Nottingham Arabidopsis Stock Centre for the da2-1 mutant. This work was supported by the National Basic Research Program of China (2009CB941503) and National Natural Science Foundation of China (31300242, 91017014, 31221063, and 30870215).
AUTHOR CONTRIBUTIONS
T.X., N.L., and Y.L. designed the research. T.X., N.L., J.L., and F.G. performed most of the experiments. N.L. and J.L. detected the E3 ubiquitin activity of DA2. J.D. and A.K. performed the DA1 ubiquitination assay. T.X., N.L., M.W.B., and Y.L. analyzed the data. T.X., N.L., and Y.L. wrote the article.
Footnotes
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.plantcell.org) is: Yunhai Li (yhli{at}genetics.ac.cn).
↵1 These authors contributed equally to this work.
↵[C] Some figures in this article are displayed in color online but in black and white in the print edition.
↵[W] Online version contains Web-only data.
Glossary
- UIM
- ubiquitin interaction motif
- DAP
- d after pollination
- GUS
- β-glucuronidase
- GST
- glutathione S-transferase
- Col-0
- Columbia-0
- Received June 18, 2013.
- Revised August 9, 2013.
- Accepted August 27, 2013.
- Published September 17, 2013.
References
