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Morphomechanical Innovation Drives Explosive Seed Dispersal

View Article: PubMed Central - PubMed

ABSTRACT

How mechanical and biological processes are coordinated across cells, tissues, and organs to produce complex traits is a key question in biology. Cardamine hirsuta, a relative of Arabidopsis thaliana, uses an explosive mechanism to disperse its seeds. We show that this trait evolved through morphomechanical innovations at different spatial scales. At the organ scale, tension within the fruit wall generates the elastic energy required for explosion. This tension is produced by differential contraction of fruit wall tissues through an active mechanism involving turgor pressure, cell geometry, and wall properties of the epidermis. Explosive release of this tension is controlled at the cellular scale by asymmetric lignin deposition within endocarp b cells—a striking pattern that is strictly associated with explosive pod shatter across the Brassicaceae plant family. By bridging these different scales, we present an integrated mechanism for explosive seed dispersal that links evolutionary novelty with complex trait innovation.

No MeSH data available.


Related in: MedlinePlus

The lig2 Mutation Prevents Nuclear Accumulation of the DNA Binding Protein LIG2, Related to Figure 2(A and B) CLSM of DAPI-stained fruit mesocarp cells from lig2; LIG2-YFP (A) and lig2; lig2-YFP (B) transgenic lines. DAPI signal (red) indicates the nucleus, YFP signal (yellow) accumulates in the nucleus in lig2; LIG2-YFP (A) cells, but is extremely reduced in lig2; lig2-YFP (B) cells, and the merged DAPI and YFP signals confirms the nuclear localization of YFP in (A).(C) RT-PCR performed on cDNA template reverse transcribed from RNA samples of lig2, lig2 LIG2-YFP and lig2 lig2-YFP transgenic lines. LIG2 and YFP primers were used to amplify a 402 bp product from the LIG2-YFP transgene and a 224 bp product from the lig2-YFP transgene. 402 bp and 224 bp amplicons in these samples indicated that both transgenes were expressed. No amplification was observed in the lig2 sample. Amplicons of the ACT8 housekeeping gene indicated equal amounts of cDNA template in each RT-PCR reaction.(D) Expression levels of LIG2, measured by qRT-PCR, are low throughout fruit development with no significant differences between stage 9 and other stages (Student’s t test p > 0.05). LIG2 is expressed in all tissues of stage 17 fruit with significantly higher expression in the seed than the valve and significantly higher expression in the valve than the rest of the fruit (Student’s t test p < 0.01). Mean values and standard deviations are shown. LIG2 expression was normalized to expression of the reference gene Clathrin/AP2M (CARHR174880).(E and F) CLSM of LIG2-YFP expression (yellow). Nuclear expression is observed in the seed and all layers of the valve in a cross section of stage 15 fruit (E). Nuclear expression is observed in the endocarp b and a layers in an en face section of a stage 16 valve; cells are outlined by propidium iodide staining (F).(G–J) Transverse valve sections, 70 μm thick, stained with phloroglucinol to visualize lignin. A lignified endocarp b cell layer is present in wild-type (G), absent in lig2 (H), restored when a LIG2-YFP transgene is introduced into the lig2 mutant (I) but not when a lig2-YFP transgene containing the lig2 mutation is introduced into the lig2 mutant (J). Scale bars: 25 μm (A, B, E), 50 μm (F), 20 μm (G-J).
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figs2: The lig2 Mutation Prevents Nuclear Accumulation of the DNA Binding Protein LIG2, Related to Figure 2(A and B) CLSM of DAPI-stained fruit mesocarp cells from lig2; LIG2-YFP (A) and lig2; lig2-YFP (B) transgenic lines. DAPI signal (red) indicates the nucleus, YFP signal (yellow) accumulates in the nucleus in lig2; LIG2-YFP (A) cells, but is extremely reduced in lig2; lig2-YFP (B) cells, and the merged DAPI and YFP signals confirms the nuclear localization of YFP in (A).(C) RT-PCR performed on cDNA template reverse transcribed from RNA samples of lig2, lig2 LIG2-YFP and lig2 lig2-YFP transgenic lines. LIG2 and YFP primers were used to amplify a 402 bp product from the LIG2-YFP transgene and a 224 bp product from the lig2-YFP transgene. 402 bp and 224 bp amplicons in these samples indicated that both transgenes were expressed. No amplification was observed in the lig2 sample. Amplicons of the ACT8 housekeeping gene indicated equal amounts of cDNA template in each RT-PCR reaction.(D) Expression levels of LIG2, measured by qRT-PCR, are low throughout fruit development with no significant differences between stage 9 and other stages (Student’s t test p > 0.05). LIG2 is expressed in all tissues of stage 17 fruit with significantly higher expression in the seed than the valve and significantly higher expression in the valve than the rest of the fruit (Student’s t test p < 0.01). Mean values and standard deviations are shown. LIG2 expression was normalized to expression of the reference gene Clathrin/AP2M (CARHR174880).(E and F) CLSM of LIG2-YFP expression (yellow). Nuclear expression is observed in the seed and all layers of the valve in a cross section of stage 15 fruit (E). Nuclear expression is observed in the endocarp b and a layers in an en face section of a stage 16 valve; cells are outlined by propidium iodide staining (F).(G–J) Transverse valve sections, 70 μm thick, stained with phloroglucinol to visualize lignin. A lignified endocarp b cell layer is present in wild-type (G), absent in lig2 (H), restored when a LIG2-YFP transgene is introduced into the lig2 mutant (I) but not when a lig2-YFP transgene containing the lig2 mutation is introduced into the lig2 mutant (J). Scale bars: 25 μm (A, B, E), 50 μm (F), 20 μm (G-J).

Mentions: To investigate whether the endocarp b layer is strictly required for explosive pod shatter, we took a genetic approach. Having shown that this stiff layer plays a mechanical role in generating valve curvature, we reasoned that an endocarp b deletion mutant should reveal how important this layer is for explosive shatter. This class of mutant had not been previously identified in A. thaliana, so rather than follow a targeted genome editing approach we conducted a mutant screen. We screened a population of ethyl methanesulfonate (EMS)-treated C. hirsuta plants for mutants with less lignified valves. In one such mutant, less lignin2 (lig2), the entire endocarp b cell layer was missing (Figures 2A–2F). We showed that lig2 is a loss-of-function mutant caused by a premature stop codon before the nuclear localization signal in the C. hirsuta ortholog of the DNA-binding protein BRASSINOSTEROID-INSENSITIVE4 (At5g24630; Figures 2G–2K) (Breuer et al., 2007, Kirik et al., 2007). LIG2 is expressed in endocarp b cells and throughout the fruit, and the lig2 mutation prevents nuclear accumulation of LIG2, resulting in loss of endocarp b layer integrity through mechanisms that remain to be determined (Figure S2). Importantly, pod shatter in the lig2 mutant was non-explosive (Figure 2D), providing genetic evidence that the endocarp b layer is indeed necessary for explosive pod shatter.


Morphomechanical Innovation Drives Explosive Seed Dispersal
The lig2 Mutation Prevents Nuclear Accumulation of the DNA Binding Protein LIG2, Related to Figure 2(A and B) CLSM of DAPI-stained fruit mesocarp cells from lig2; LIG2-YFP (A) and lig2; lig2-YFP (B) transgenic lines. DAPI signal (red) indicates the nucleus, YFP signal (yellow) accumulates in the nucleus in lig2; LIG2-YFP (A) cells, but is extremely reduced in lig2; lig2-YFP (B) cells, and the merged DAPI and YFP signals confirms the nuclear localization of YFP in (A).(C) RT-PCR performed on cDNA template reverse transcribed from RNA samples of lig2, lig2 LIG2-YFP and lig2 lig2-YFP transgenic lines. LIG2 and YFP primers were used to amplify a 402 bp product from the LIG2-YFP transgene and a 224 bp product from the lig2-YFP transgene. 402 bp and 224 bp amplicons in these samples indicated that both transgenes were expressed. No amplification was observed in the lig2 sample. Amplicons of the ACT8 housekeeping gene indicated equal amounts of cDNA template in each RT-PCR reaction.(D) Expression levels of LIG2, measured by qRT-PCR, are low throughout fruit development with no significant differences between stage 9 and other stages (Student’s t test p > 0.05). LIG2 is expressed in all tissues of stage 17 fruit with significantly higher expression in the seed than the valve and significantly higher expression in the valve than the rest of the fruit (Student’s t test p < 0.01). Mean values and standard deviations are shown. LIG2 expression was normalized to expression of the reference gene Clathrin/AP2M (CARHR174880).(E and F) CLSM of LIG2-YFP expression (yellow). Nuclear expression is observed in the seed and all layers of the valve in a cross section of stage 15 fruit (E). Nuclear expression is observed in the endocarp b and a layers in an en face section of a stage 16 valve; cells are outlined by propidium iodide staining (F).(G–J) Transverse valve sections, 70 μm thick, stained with phloroglucinol to visualize lignin. A lignified endocarp b cell layer is present in wild-type (G), absent in lig2 (H), restored when a LIG2-YFP transgene is introduced into the lig2 mutant (I) but not when a lig2-YFP transgene containing the lig2 mutation is introduced into the lig2 mutant (J). Scale bars: 25 μm (A, B, E), 50 μm (F), 20 μm (G-J).
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figs2: The lig2 Mutation Prevents Nuclear Accumulation of the DNA Binding Protein LIG2, Related to Figure 2(A and B) CLSM of DAPI-stained fruit mesocarp cells from lig2; LIG2-YFP (A) and lig2; lig2-YFP (B) transgenic lines. DAPI signal (red) indicates the nucleus, YFP signal (yellow) accumulates in the nucleus in lig2; LIG2-YFP (A) cells, but is extremely reduced in lig2; lig2-YFP (B) cells, and the merged DAPI and YFP signals confirms the nuclear localization of YFP in (A).(C) RT-PCR performed on cDNA template reverse transcribed from RNA samples of lig2, lig2 LIG2-YFP and lig2 lig2-YFP transgenic lines. LIG2 and YFP primers were used to amplify a 402 bp product from the LIG2-YFP transgene and a 224 bp product from the lig2-YFP transgene. 402 bp and 224 bp amplicons in these samples indicated that both transgenes were expressed. No amplification was observed in the lig2 sample. Amplicons of the ACT8 housekeeping gene indicated equal amounts of cDNA template in each RT-PCR reaction.(D) Expression levels of LIG2, measured by qRT-PCR, are low throughout fruit development with no significant differences between stage 9 and other stages (Student’s t test p > 0.05). LIG2 is expressed in all tissues of stage 17 fruit with significantly higher expression in the seed than the valve and significantly higher expression in the valve than the rest of the fruit (Student’s t test p < 0.01). Mean values and standard deviations are shown. LIG2 expression was normalized to expression of the reference gene Clathrin/AP2M (CARHR174880).(E and F) CLSM of LIG2-YFP expression (yellow). Nuclear expression is observed in the seed and all layers of the valve in a cross section of stage 15 fruit (E). Nuclear expression is observed in the endocarp b and a layers in an en face section of a stage 16 valve; cells are outlined by propidium iodide staining (F).(G–J) Transverse valve sections, 70 μm thick, stained with phloroglucinol to visualize lignin. A lignified endocarp b cell layer is present in wild-type (G), absent in lig2 (H), restored when a LIG2-YFP transgene is introduced into the lig2 mutant (I) but not when a lig2-YFP transgene containing the lig2 mutation is introduced into the lig2 mutant (J). Scale bars: 25 μm (A, B, E), 50 μm (F), 20 μm (G-J).
Mentions: To investigate whether the endocarp b layer is strictly required for explosive pod shatter, we took a genetic approach. Having shown that this stiff layer plays a mechanical role in generating valve curvature, we reasoned that an endocarp b deletion mutant should reveal how important this layer is for explosive shatter. This class of mutant had not been previously identified in A. thaliana, so rather than follow a targeted genome editing approach we conducted a mutant screen. We screened a population of ethyl methanesulfonate (EMS)-treated C. hirsuta plants for mutants with less lignified valves. In one such mutant, less lignin2 (lig2), the entire endocarp b cell layer was missing (Figures 2A–2F). We showed that lig2 is a loss-of-function mutant caused by a premature stop codon before the nuclear localization signal in the C. hirsuta ortholog of the DNA-binding protein BRASSINOSTEROID-INSENSITIVE4 (At5g24630; Figures 2G–2K) (Breuer et al., 2007, Kirik et al., 2007). LIG2 is expressed in endocarp b cells and throughout the fruit, and the lig2 mutation prevents nuclear accumulation of LIG2, resulting in loss of endocarp b layer integrity through mechanisms that remain to be determined (Figure S2). Importantly, pod shatter in the lig2 mutant was non-explosive (Figure 2D), providing genetic evidence that the endocarp b layer is indeed necessary for explosive pod shatter.

View Article: PubMed Central - PubMed

ABSTRACT

How mechanical and biological processes are coordinated across cells, tissues, and organs to produce complex traits is a key question in biology. Cardamine hirsuta, a relative of Arabidopsis thaliana, uses an explosive mechanism to disperse its seeds. We show that this trait evolved through morphomechanical innovations at different spatial scales. At the organ scale, tension within the fruit wall generates the elastic energy required for explosion. This tension is produced by differential contraction of fruit wall tissues through an active mechanism involving turgor&nbsp;pressure, cell geometry, and wall properties of the epidermis. Explosive release of this tension is&nbsp;controlled at the cellular scale by asymmetric lignin&nbsp;deposition within endocarp b cells&mdash;a striking pattern that is strictly associated with explosive pod shatter across the Brassicaceae plant family. By bridging these different scales, we present an integrated mechanism for explosive seed dispersal that links evolutionary novelty with complex trait innovation.

No MeSH data available.


Related in: MedlinePlus