The MADS Box Genes <i>ABS</i>, <i>SHP1</i>, and <i>SHP2</i> Are Essential for the Coordination of Cell Divisions in Ovule and Seed Coat Development and for Endosperm Formation in <i>Arabidopsis thaliana</i>

<div><p>Seed formation is a pivotal process in plant reproduction and dispersal. It begins with megagametophyte development in the ovule, followed by fertilization and subsequently coordinated development of embryo, endosperm, and maternal seed coat. Two closely related MADS-box genes, <i>SHATTERPROOF 1</i> and <i>2</i> (<i>SHP1</i> and <i>SHP2</i>) are involved in specifying ovule integument identity in <i>Arabidopsis thaliana</i>. The MADS box gene <i>ARABIDOPSIS BSISTER</i> (<i>ABS</i> or <i>TT16</i>) is required, together with <i>SEEDSTICK</i> (<i>STK</i>) for the formation of endothelium, part of the seed coat and innermost tissue layer formed by the maternal plant. Little is known about the genetic interaction of <i>SHP1</i> and <i>SHP2</i> with <i>ABS</i> and the coordination of endosperm and seed coat development. In this work, mutant and expression analysis shed light on this aspect of concerted development. Triple <i>tt16 shp1 shp2</i> mutants produce malformed seedlings, seed coat formation defects, fewer seeds, and mucilage reduction. While <i>shp1 shp2</i> mutants fail to coordinate the timely development of ovules, <i>tt16</i> mutants show less peripheral endosperm after fertilization. Failure in coordinated division of the innermost integument layer in early ovule stages leads to inner seed coat defects in <i>tt16</i> and <i>tt16 shp1 shp2</i> triple mutant seeds. An antagonistic action of <i>ABS</i> and <i>SHP1/SHP2</i> is observed in inner seed coat layer formation. Expression analysis also indicates that <i>ABS</i> represses <i>SHP1</i>, <i>SHP2</i>, and <i>FRUITFUL</i> expression. Our work shows that the evolutionary conserved B_sister genes are required not only for endothelium but also for endosperm development and genetically interact with <i>SHP1</i> and <i>SHP2</i> in a partially antagonistic manner.</p></div

Quantitative analysis of the presence of the ii1´ seed coat layer in well-developed seeds of stage 16 and 17A siliques harboring globular to heart staged embryos.

<p>(A) and (B), percentages of seeds with, without, and with only partially developed ii1´ layer, or with a locally occurring additional fourth layer on the chalazal side (A) and on the micropylar side (B). For Ws-4, n = 124; Col-0, n = 97; <i>tt16</i>, n = 151; <i>shp1 shp2</i>, n = 94; <i>tt16 shp1 shp2</i>, n = 57. (C)-(J), documentation of the seed coat layer anatomy at the chalazal side of well-developed seeds in the wild type and mutant lines. In Ws-4 (C) and Col-0 (D) wild type seeds, the ii1´ cell layer usually is complete and reaches up to the chalazal end (additional incomplete fourth cell layer is formed in some cells, between arrowheads in C). ii1 and ii1´ cells have an almost isodiametric shape (C, D), with ii1´ cells being highly vacuolated whereas the ii1 cells (endothelium) appear densely packed. The <i>shp1 shp2</i> seed coat may show an incomplete, extra layer of thick, vacuolated cells adjacent to the ii1´ layer (between arrowheads in E) or the normal five layers (F). (G), <i>tt16</i> seed coat with five complete cell layers showing thin elongated cells in the inner two layers (cf. also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165075#pone.0165075.s005" target="_blank">S3F Fig</a>). (H), <i>tt16</i> seed coat, partially missing the ii1´ layer which does not reach the chalazal end (below arrowhead) and (I), complete lack of the ii1´ layer at the chalazal side of a <i>tt16</i> seed coat. (J), <i>tt16 shp1 shp2</i> mutant seed coat with five complete cell layers but the two inner seed coat layers are composed of thin elongated cells lacking dense cytoplasm. Scale bars in (C)-(J) are 50 μm.</p

Relative expression of the MADS box genes <i>ABS</i>, <i>SHP1</i>, <i>SHP2</i>, <i>AGL15</i>, <i>FUL</i>, and <i>STK</i> in Ws-4, Col-0, <i>tt16</i>, <i>shp1 shp2</i>, and <i>tt16 shp1 shp2</i> analyzed by qRT-PCR.

<p>(A), gene expression in floral buds, (B), expression in siliques of stage 17A. Bars indicate standard deviations, stars indicate statistically significant difference (p < 0.05, tested by the one-tailed t-test) in expression, expression in <i>tt16</i> plants was compared to Ws-4, expression in <i>shp1 shp2</i> was compared to Col-0 and expression in <i>tt16 shp1 shp2</i> was compared to both wild types. (C), simplified model of genetic interactions between <i>ABS</i>, <i>SHP1</i>, <i>SHP2</i>, <i>FUL</i>, and <i>STK</i> (this work and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165075#pone.0165075.ref045" target="_blank">45</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165075#pone.0165075.ref052" target="_blank">52</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165075#pone.0165075.ref053" target="_blank">53</a>]). A blue line indicates genetic interaction in floral buds, an orange line indicates regulation at stage 17A, a black line regulation at both stages of development, and a grey line indicates a gene´s impact on a morphological process. X symbolizes the temporal gap between expression and phenotypical effect.</p

Seed developmental defects of Ws-4 and Col-0 wild type plants and of <i>tt16</i>, <i>shp1 shp2</i>, and <i>tt16 shp1 shp2</i> mutants.

<p>(A), amount of unfertilized ovules/very young seeds (classified as stage 3-VI of megagametogenesis or post-fertilization stage 4-I according to Schneitz et al., 1995 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165075#pone.0165075.ref044" target="_blank">44</a>]), ovules/seeds with developmental defects, and properly developed seeds given in percentages of the total number of ovules/seeds found in stage 16 and stage 17A siliques (stages defined according to Ferrándiz et al., 1999 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165075#pone.0165075.ref045" target="_blank">45</a>]) containing octant, globular or early heart embryos. For Ws-4, n = 257; Col-0, n = 193; <i>tt16</i>, n = 463; <i>shp1 shp2</i>, n = 234; <i>tt16 shp1 shp2</i>, n = 202. (B), properly developed seed of a Ws-4 plant with a globular embryo. The micropylar and chalazal endosperm appears intact (arrowheads) and the syncytial peripheral endosperm contains many nuclei (e.g. circles). The seed coat develops from the two layers of the outer integument (oi2 and oi1) and the three layers of the inner integument (ii2, ii1´, and ii1). The innermost endothelium layer (ii1) is composed of thick cells with an almost isodiametric shape. Properly developed seeds of Col-0 plants appear similar. (C) to (K), unfertilized ovules/very young seeds and malformed seeds found in stage 16 and stage 17A siliques harboring octant, globular or early heart embryos. Developmental defects in Ws-4 (C) or Col-0 (D) where the lack of a properly developed embryo and shrinkage of the integument or seed coat layers (between arrowheads) indicates that these ovules/seeds are most likely destined to abort. (E), Col-0, rare case of an unfertilized ovule/very young seed with a normal appearance. (F) and (G), small-sized and slender <i>tt16</i> mutant seeds harboring embryos at globular (F) and early heart stage (G), but containing only little (G, circles) or no peripheral endosperm (F). The embryo in (F) appears mechanically squeezed, possibly due to its short suspensor and the small seed size. (H) shows an unfertilized ovule of normal appearance which were regularly found next to well-developed seeds in the siliques of <i>shp1 shp2</i> mutant plants. AC: antipodal cell; CC: central cell nucleus; EC: egg cell nucleus. (I), a presumably unfertilized and aborted ovule of a <i>tt16 shp1 shp2</i> triple mutant shows a malformed embryo sac (black arrow) and shrunken integument layers. (J), small-sized <i>tt16 shp1 shp2</i> seed containing an early globular embryo with a slight defect in the protoderm, but no endosperm. (K), slender seed of a <i>tt16 shp1 shp2</i> plant with well-developed micropylar endosperm surrounding the early heart stage embryo, but only few peripheral endosperm nuclei / cells directly attached to the seed coat (circles). This seed resembles the seed of the <i>tt16</i> mutant in (G). Scale bars in (B)-(K) are 50 μm.</p

Ovule anatomy of Ws-4 wild type plants and developmental defects of <i>tt16</i>, <i>shp1 shp2</i>, and <i>tt16 shp1 shp2</i> mutant ovules of floral stage 12 (stages defined after Smyth et al., 1990) which were classified as almost mature and fully mature ovules at late stage 3-V or stage 3-VI (stages defined after Schneitz et al., 1995 [44]).

<p>(A)-(C), Ws-4 ovules, DIC micrograph (A) and colorized version of the same image (B) showing ovule at stage 3-V whose eight-nuclear embryo sac (yellow dots) has undergone cellularization. Labels are as follows: outer integument 2 and 1 (oi2, dark red; oi1, light red), inner integument 2, 1´, and 1 (ii2, dark green; ii1´, green; ii1, light green); ad, adaxial side; ab, abaxial side; double headed arrow, curving zone; mi, micropyle; nu, nucellus; star, chalazal end). Ovule anatomy is similar in Ws-4 and Col-0. (C), CLSM micrograph of a Ws-4 ovule stained with propidium iodide also illustrates the normal occurrence of the ii1´ layer at the adaxial (between yellow arrowheads) and abaxial side (between white arrowheads). For the abaxial side, the start of the ii1´ layer directly at the chalazal end (star) and the end of the ii1´ layer are shown enlarged in the respective insert (white arrowheads). (D), normal development of the abaxial ii1´ layer of a <i>tt16</i> mutant ovule (between arrowheads). (E), <i>tt16</i> ovule, where several cells at the chalazal end (star) of the abaxial ii1 layer fail to undergo periclinal divisions, but formation of the ii1´ layer (between arrowheads and see insert) starts further down towards the micropylar end. (F), patchy development of the ii1´ layer occurring at the abaxial side of <i>tt16</i> mutant ovules. Periclinal divisions start at two independent positions (between white arrowheads, enlarged in the inserts). (G), normal development of the ii1´ layer (between arrowheads) at the abaxial side of a <i>shp1 shp2</i> ovule which contains an almost mature embryo sac. (H), young ovule at an earlier stage of megagametogenesis (3-IV, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165075#pone.0165075.ref044" target="_blank">44</a>]) found besides almost mature and fully mature ovules in the same <i>shp1 shp2</i> pistil. The nuclei of the four-nuclear embryo sac are marked with yellow circles. (I), normal development of the abaxial ii1´ layer of a <i>tt16 shp1 shp2</i> ovule (between white arrowheads). At the adaxial side, the first two ii1´ cells formed by periclinal divisions of ii1 at the chalazal end (between yellow arrowheads). (J), the formation of the ii1´ layer at the abaxial side of this <i>tt16 shp1 shp2</i> ovule fails to start at the chalazal end (star), but starts a few cells further down towards the micropylar end (between arrowheads). (K) and (L), development of some <i>tt16 shp1 shp2</i> ovules also lags behind in comparison to other ovules of the same pistil. Formation of the abaxial ii1´ layer has only recently begun and only one or two ii1 cells have already divided at the chalazal end (between arrowheads in Figs (K) and (L), and shown enlarged in the insert of (L)). The <i>tt16 shp1 shp2</i> ovule shown in (L) is at an earlier stage of megagametogenesis than the almost mature ovules found in the same pistil. Scale bars in (A)-(L) are 50 μm. Clearing of the propidium-iodide stained CLSM samples was performed with chloral hydrate in (C) and (L), and with benzyl benzoate/benzyl alcohol in (E) and (F). Ovules of at least five pistils collected from different plants were observed for every plant line.</p

Defects in seedling development, mucilage release, and seed oil content analysis in Ws-4 and Col-0 wild type plants and in <i>tt16</i>, <i>shp1 shp2</i>, and <i>tt16 shp1 shp2</i> mutants.

<p>(A), cotyledon greening assay of seedlings grown on soil with appearance of green cotyledons scored as criterion for the viability. (B), proportion of seedlings germinated on soil without observed developmental defects, star indicates significant difference to Ws-4 (student’s t-test, p ≤ 0.05). Seed morphology is shown of Ws-4 (C), Col-0 (D), <i>tt16</i> (E), <i>shp1 shp2</i> (F), and <i>tt16 shp1 shp2</i> (G) seeds. Arrowheads in (E) and (G) indicate malformed seeds. Ruthenium Red assay was carried out to observe the quantity and distribution of mucilage on hydrated seeds of Ws-4 (H), Col-0 (I), <i>tt16</i> (J), <i>shp1 shp2</i> (K), and <i>tt16 shp1 shp2</i> (L) seeds. (M)-(R), one week old seedlings of the Col-0 (M), and <i>tt16 shp1 shp2</i> mutants germinated on soil (N)-(R) illustrating the different types of abnormal seedling development observed, such as short roots, fused cotyledons, malformed hypocotyls, and a reduction in size of the cotyledons. (S), comparison of mature seed fatty acid composition between wild type (WT) <i>A</i>. <i>thaliana</i> accessions (Col-0, Ws-4) and the mutant genotypes <i>tt16</i>, <i>shp1 shp2</i> and <i>tt16 shp1 shp2</i>, respectively. Significances were tested with a Post-Hoc-test. Circles: p<0.001 when compared to all other plant lines; stars: p<0.0001 when compared to all other plant lines; arrowheads: p<0.0001 when compared to the wild types or to the <i>shp1 shp2</i> mutant. Scale bars in C, D, E, F, G, N: 500μm; H, I, J, K, L: 200μm; M: 2mm; O, P, Q, R: 1mm.</p

GUS staining assay of pABS::ABS:GUS.

<p>Young ovules in buds of stage 10 (A), in unfertilized ovules in stage 13 flowers (B), and in seeds from siliques at stage 16 to 17A harboring globular embryos (C) and heart stage embryos (D) were analyzed. Green arrowhead: staining in the nucellus and inner integument; black arrowheads: micropylar end; red arrowheads: chalazal endosperm; white arrowheads show the egg cell (B) and embryo (C, D); Scale bars: 50μm.</p