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Negative Regulation of the SHATTERPROOF Genes by FRUITFULL During Arabidopsis Fruit Development

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Science  21 Jul 2000:
Vol. 289, Issue 5478, pp. 436-438
DOI: 10.1126/science.289.5478.436

Abstract

The terminal step of fruit development in Arabidopsisinvolves valve separation from the replum, allowing seed dispersal. This process requires the activities of theSHATTERPROOF MADS-box genes, which promote dehiscence zone differentiation at the valve/replum boundary. Here we show that the FRUITFULL MADS-box gene, which is necessary for fruit valve differentiation, is a negative regulator ofSHATTERPROOF expression and that constitutive expression of FRUITFULL is sufficient to prevent formation of the dehiscence zone. Our studies suggest that ectopic expression ofFRUITFULL may directly allow the control of pod shatter in oilseed crops such as canola.

The fruit mediates the maturation and dispersal of seeds and is derived from the female reproductive structure, the gynoecium. The Arabidopsis fruit, which is typical of more than 3000 species of Brassicaceae, consists of an apical stigma, a short style, and a basal ovary that contains the developing seeds (Fig. 1A). The peripheral walls of the fruit are referred to as valves and are connected on both sides by a thin structure known as the replum. At the valve/replum boundary, a narrow band of cells differentiates into the dehiscence zone (1), where the separation of cells late in fruit development allows valve detachment from the replum and seed dispersal.

Figure 1

Constitutive expression of FUL converts cells of the valve margin and outer replum into valve cells. Scanning electron micrographs (A and B) and transverse sections of fruit stained with toluidine blue (C andD) or phloroglucinol (E to G) at stage 17 (A to F) or stage 18 (G). In wild-type fruit (A, C, and E), cells at the valve margin differentiate into the dehiscence zone (DZ), and lignification of the valve inner subepidermal layer (lv) and small patches of valve margin cells (vm) adjacent to the dehiscence zone occurs. 35S::FUL fruit (B and D) have reduced styles (sty) and appear radially uniform, as cells within the valve margin and outer replum (r) regions closely resemble wild-type valve (v) cells. Because of these cell fate conversions, dehiscence zone differentiation and valve margin lignification do not occur in 35S::FUL fruit (D and F). In contrast, ful fruit (G) show ectopic lignification of the valve mesophyll (me) layers; stg, stigma; gu, guard cells; vb, vascular bundle. Bars, 100 μm.

Because FRUITFULL (FUL) is required for the expansion and differentiation of fruit valves after fertilization (2), we generated transgenic plants in which FULis constitutively expressed from the cauliflower mosaic virus 35S promoter (3–5) to determine ifFUL is sufficient to specify valve cell fate in ectopic positions. The most striking phenotype caused by the 35S::FUL transgene is the conversion of cells within the valve margin and outer replum to valve cells (Fig. 1). Consequently, the dehiscence zone, which normally forms at the valve margin (Fig. 1, A and C), fails to differentiate in 35S::FUL fruit (Fig. 1, B and D). Thus, likeshatterproof (shp1 shp2) loss-of-function mutants (6), 35S::FUL gain-of-function plants produce indehiscent fruit and fail to disperse their seeds normally.

Because lignification is thought to play an important role in the dehiscence process (1), and because the SHP genes promote lignification of cells adjacent to the dehiscence zone (6), we examined the lignification patterns of 35S::FUL and ful fruit compared with that seen in the wild type (7). Whereas only a single valve cell layer is lignified in wild-type fruit (Fig. 1E), all of the internal valve mesophyll layers also become lignified in ful fruit (Fig. 1G). Correspondingly, in 35S::FUL fruit (Fig. 1F), we found a consistent loss of lignified cells adjacent to where the dehiscence zone normally forms. Therefore, FUL is necessary to prevent ectopic lignification of certain valve cells, and constitutive expression of FUL is sufficient to prevent valve margin lignification.

The similarities between the indehiscent phenotypes of 35S::FUL and shp1 shp2 fruit, together with the complementary expression patterns of FUL and SHP(3, 8, 9), suggest either thatFUL negatively regulates SHP1/2, thatSHP1/2 negatively regulate FUL, or thatFUL and SHP1/2 repress each other. To determine if FUL is a negative regulator of the SHP genes, we compared the accumulation of SHP RNAs in wild-type,ful, and 35S::FUL fruit (10). We observed that the domains of SHP1 (Fig. 2, A and B) and SHP2(11) expression expanded throughout the valves offul fruit, and conversely, that SHP expression was down-regulated in 35S::FUL fruit (Fig. 2, C and D). Thus, FUL is a negative spatial regulator of SHP RNA accumulation. To determine if the SHP gene products negatively regulateFUL, we analyzed the expression pattern of FUL in wild-type, shp1 shp2, and 35S::SHP1/2 fruit (10). We found that the domain of FUL expression did not expand to include the valve margin regions of shp1 shp2 fruit (Fig. 2, E and F), and that expression of the FUL::GUS marker was not down-regulated in 35S::SHP1/2 fruit (Fig. 2, G and H). Therefore, SHP1/2 do not negatively regulate FUL RNA accumulation.

Figure 2

FUL negatively regulates SHP and GT140 expression. Expression of SHP1 (A toC), SHP2 (D), FUL(E to H), and the GT140 valve margin marker (I to L) was analyzed by in situ hybridization (A to F) or by GUS reporter detection (G to L) in transverse sections of developing fruit at stage 13 (E and F), stage 15 (A to D), stage 16 (G to I and K), and stage 17 (J and L). In wild-type fruit,SHP1 (A), SHP2 (11), and GT140 (I) are expressed in stripes at the valve margin (vm) where the dehiscence zone will form. Expression of SHP1, SHP2, and GT140 expands throughout the valves (v) of ful-2 fruit [B, J, and (11)] and is not detected in 35S::FUL fruit (C, D, and K). In shp1 shp2 ful-2 fruit (L), expression of GT140 is detected throughout the valves at reduced levels. FUL is expressed in wild-type fruit valves (E) and does not expand beyond the valves of shp1 shp2 fruit (F). In 35S::SHP1 35S::SHP2 ful-1/+ fruit (H), FUL::GUS expression does not appear reduced as compared with that seen in ful-1/+ fruit valves (G). Bars, 100 μm.

The observation that SHP1/2 are ectopically expressed throughout the valves of ful mutants suggested that some of the ful mutant phenotypes could be due to ectopicSHP activity. To investigate this possibility, we comparedshp1 shp2 ful fruit (12) to ful fruit (Fig. 3). While they appeared very similar (Fig. 3A),shp1 shp2 ful fruit exhibited tears in the valves due to seed crowding (Fig. 3, B and C) less frequently, had guard cells in the valve outer epidermis (Fig. 3, D to F), and were indehiscent. AlthoughSHP activity promotes valve margin lignification (6), the valves of shp1 shp2 ful fruit are still ectopically lignified (11), indicating that ectopic SHP activity is not primarily responsible for this and other ful mutant phenotypes.

Figure 3

Mutations in SHP1 and SHP2partially suppress the valve differentiation defects of fulfruit and largely eliminate valve tearing. (A) Likeful-1 fruit, shp1 shp2 ful-1 fruit (stage 17) are much shorter than the wild type, due to the lack of valve expansion after fertilization. Valve tears (arrowhead) are present in nearly allful-1 fruit (B) and are rarely seen in shp1 shp2 ful-1 fruit (C). Guard cells (gu) are present in wild-type and shp1 shp2 ful-1 fruit valves (D andF) and are not found in ful-1 fruit valves (E). Bars, 100 μm.

To identify additional genes regulated by FUL that may contribute to ful phenotypes, we analyzed expression of the valve margin marker GT140 (13, 14) inful and 35S::FUL fruit compared with expression in the wild type. Whereas GT140 is normally expressed in narrow stripes at the valve margin (Fig. 2I), this marker was ectopically expressed throughout ful mutant valves (Fig. 2J) and was largely absent in 35S::FUL fruit (Fig. 2K). Because SHPactivity is required for GT140 valve margin expression (6), and SHP expression also expands throughout ful valves and is down-regulated in 35S::FUL fruit, ectopic GT140 expression in fulfruit could be entirely due to ectopic SHP activity. To investigate this possibility, we also analyzed expression of the GT140 marker in shp1 shp2 ful fruit. GT140 was still expressed throughout the triple-mutant valves (Fig. 2L), although at a reduced level relative to that seen in ful valves (Fig. 2J). These data suggest that FUL negatively regulates GT140 expression and demonstrate that SHP activity is not absolutely required for GT140 expression in ful mutant valves, indicating that additional genes are involved in activating this marker.

The data presented here, together with other recently published observations, allow us to propose a model (Fig. 4) for some of the genetic interactions underlying valve margin development. The SHP genes are positively regulated by the AGAMOUS MADS-box gene product (8, 9) and are required for proper valve margin development (6). Besides directing valve differentiation (2), FUL negatively regulates SHPexpression, ensuring that valve margin cell fate occurs only at the valve boundary. Although not shown in the model, SHP1/2 may negatively regulate a replum-specific factor; expansion of such a factor's activity in shp1 shp2 fruit could account for the observed slight restriction of FUL valve expression (Fig. 2, E and F). Expression of the GT140 valve margin marker is positively regulated by SHP1/2 (6) and negatively regulated by FUL, which may occur by way of an additional factor (factor X) involved in GT140 activation.

Figure 4

Genetic interactions involved in valve margin development.

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: marty{at}ucsd.edu

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