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Overaccumulation of Suc induces wild-type epidermal cells to express the tdy1 mutant phenotype. A, C, E, G, and I, Bright-field images of the abaxial epidermis in wild-type and mosaic sectors. Insets indicate genotype of TLs in the transverse dimension determined by free-hand cross section of the tissue. B, D, F, H, and J, Same images viewed under blue light showing presence or absence of chlorophyll autofluorescence in guard cells. Arrows in B and J indicate chlorophyll autofluorescence in guard cells. A and B, Wild-type tissue showing red chlorophyll autofluorescence. C and D, w14 tdy1 / 2 albino sector expressing the tdy1 anthocyanin accumulation phenotype. No chlorophyll autofluorescence is observed in the guard cells, indicating the epidermis is genetically mutant. E and F, Wild-type epidermis overlying
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The tie-dyed1 (tdy1) mutant of maize (Zea mays) produces chlorotic, anthocyanin-accumulating regions in leaves due to the hyperaccumulation of carbohydrates. Based on the nonclonal pattern, we propose that the accumulation of sucrose (Suc) or another sugar induces the tdy1 phenotype. The boundaries of regions expressing the tdy1 phenotype frequentl...
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Context 1
... Mutator trans- posable element (Fig. 4A). In the absence of Mutator activity, Yg - str* mutants are pale-green seedling le- thals, indicating that the mutation abrogates chloroplast function (D. Braun, unpublished data). In Yg - str*; tdy1 double mutant leaves, tdy1 phenotypic regions are indicated by the accumulation of anthocyanin. We found that these regions localize to the revertant green yg tissue (Fig. 4B). The only exceptions to this observation occurred if a tdy1 region was sufficiently large (Fig. 4B, arrows). To verify the cellular expression of the tdy1 phenotype, we examined tissues from Yg - str*; tdy1 double mutant leaves for chlorophyll and starch accumulation (Fig. 4, C–N). Green tissue in revertant yg sectors displays greater chlorophyll levels than in tdy1 phenotypic regions (Fig. 4, C, D, F, and G). Additionally, tdy1 regions in revertant yg tissue accumulate anthocyanin and starch, demonstrating that yg revertant tissue expresses all aspects of the tdy1 phenotype (Fig. 4, E, F, and H). Yg - str* pale-green mutant tissue displays reduced chlorophyll abundance (Fig. 4I) yet increased chlorophyll autofluorescence (Fig. 4J) similar to some high-chlorophyll fluorescence mutants (Miles and Daniel, 1974). Yg - str* pale-green mutant tissues do not accumulate starch (Fig. 4K). Occasionally, a tdy1 region spread into adjacent Yg - str* pale- green mutant tissue (Fig. 4, B, arrows, and L), and the chlorophyll autofluorescence seen in pale-green mutant tissue was reduced (compare Fig. 4, J and M). These tissues expressed anthocyanin and exhibited starch accumulation in mesophyll cells similar to tdy1 regions in yg revertant tissue (Fig. 4, H and N). As tdy1 regions only initiate within yg revertant tissue, these data suggest functional chloroplasts are needed to generate the Suc that overaccumulates and induces tdy1 phenotypic expression. Additionally, if sufficient levels accumulate, Suc can move into neighboring pale-green mutant cells, causing them to express the tdy1 phenotype (Fig. 4, B and L–N), as seen in ij1; tdy1 plants. As chloroplasts are needed to generate the sugars that induce tdy1 phenotypic expression, one possibility is that Tdy1 functions in photosynthetic cells. To test this hypothesis and determine the cell autonomy of Tdy1 , we performed a clonal mosaic analysis. Based on the ij1; tdy1 double mutant studies, we knew that tdy1 regions can be detected in albino tissues by anthocyanin and starch accumulation (Fig. 3). Genetic stocks were constructed in which tdy1 was linked in coupling to the proximally located albino mutant white14 ( w14 ), and the homologous chromosome carried wild-type functional alleles of both genes (Fig. 5A; Supplemental Fig. S1). g irradiation of germinating seeds induced chromosome breakage and uncovered albino, aneuploid w14 tdy1 / 2 sectors present in otherwise wild- type green plants. We analyzed white tissues marked by anthocyanin pigmentation to determine which cell layers lacked wild-type Tdy1 function and thereby resulted in expression of the tdy1 phenotype (Fig. 5B). In this experiment, we observed tdy1 regions within albino tissue (Figs. 5B and 8A; Supplemental Fig. S2). This suggests that wild-type cells containing functional chloroplasts produce the Suc that moves into adjoining mutant tissue, in which the absence of TDY1 results in Suc overaccumulation and a mutant phenotype. For describing tissue layers (TLs) in the transverse dimension, we utilized the numbering system of Nelson et al. (2002; Fig. 5D). In brief, adaxial and abaxial epidermal layers are respectively termed TL1 and TL5, subepidermal mesophyll layers TL2 and TL4, and the innermost layer, containing the veins, bundle sheath cells, and interveinal mesophyll cells, is termed TL3. Chimeric sectors analyzed in the mosaic analysis are summarized in Figure 5E and Supplemental Table S1. Ten genotypic classes (i–x) of chimeric sectors expressed a tdy1 phenotype. In examining the distribution of tdy1 regions within mosaic leaves, there was no effect whether the albino sectors were located on lower (juvenile) or upper (adult) leaves, nor were differences found between a single albino sector on an isolated leaf or on a meristematic sector encompassing multiple leaves. Likewise, sector position within the lateral dimension of the leaf had no effect on tdy1 phenotypic expression (Supplemental Table S1). Examination of w14 hemizygous tissue from control plants not carrying tdy1 showed aneuploidy for chromosome 6 did not result in anthocyanin deposition (Fig. 5C) or produce any adverse affects on leaf development, as previously reported (Walker and Smith, 2002). To genetically dissect the site and mode of Tdy1 function, we investigated whether a wild-type epidermis prevented expression of the mutant phenotype. For each albino sector, the epidermal genotype was determined by examining guard cells for chlorophyll autofluorescence, as these are the only epidermal cells containing chloroplasts (Fig. 6, A and B). In wild-type tissue, guard cell chloroplasts can be seen under blue light as two bright red points within each guard cell pair (Fig. 6, B, F, H, and J). Free-hand transverse cross sections of the tissue were inspected to determine the genotype of internal TLs. In entirely albino tissues expressing the tdy1 phenotype (Fig. 6C), chlorophyll autofluorescence is absent from guard cells (Figs. 5E [class i] and 6D). However, we identified multiple sectors in which a genetically wild-type epidermal layer(s) overlaid entirely mutant internal layers and exhibited a tdy1 phenotype, as marked by anthocyanin accumulation (Figs. 5E [classes iv–vi] and 6, E and F). Consistent with this, we observed an epidermal meri- clinal sector where wild-type and mutant tissues are juxtaposed, and both strongly display the tdy1 anthocyanin-accumulating phenotype regardless of their genotype (Fig. 6, G and H). Other periclinal chimeric sectors with a wild-type epidermal layer and different internal layers composed of genetically wild-type or mutant layers also expressed a tdy1 mutant phenotype (Fig. 5E, classes vii–x). For instance, in a class x chimeric sector, despite both epidermal layers and one of the internal layers being wild type, the epidermal layers expressed a tdy1 phenotype (Figs. 5E and 6, I and J). In every case examined, wild-type Tdy1 function in the epidermis was not sufficient to prevent a tdy1 region from forming. Thus, independent of its genotype, the epidermis phenotypically reflected the phenotype of the internal layers. These data also demonstrate that genotypically wild-type Tdy1 epidermal cells can be induced to express the tdy1 mutant phenotype, presumably due to accumulation and movement of Suc from underlying mutant cells. To investigate in which internal TLs Tdy1 functions and to determine its cell autonomy, aneuploid w14 tdy1 / 2 chimeric sectored leaves expressing tdy1 regions were examined. As shown previously, wild-type tissue displays abundant chlorophyll levels and shows starch accumulation only in bundle sheath cells (Fig. 7, A–D). White w14 tdy1 / 2 sectors not expressing anthocyanin lacked both chlorophyll and starch (Fig. 7, E–H). In contrast, w14 tdy1 / 2 albino sectors containing tdy1 phenotypic regions marked by anthocyanin lacked chlorophyll (Fig. 7, I and J) and accumulated starch in both mesophyll and bundle sheath cells (Fig. 7, K and L). These results show aneuploid leaf sectors express the tdy1 phenotype. In periclinal chimeras, tdy1 regions were observed only in instances where the TL3 was mutant (Fig. 5E [classes ii–x]; Supplemental Table S1). This can be seen in an example where part of the TL4 was genetically wild type, as evidenced by the presence of chlorophyll, but exhibited the diminished chlorophyll abundance of a tdy1 region (Fig. 7, M and N; compare with Figs. 2B and 7B). Moreover, the TL4 mesophyll cells accumulated starch, which is a hallmark of tdy1 phenotypic expression (Fig. 7, O and P). Hence, internal wild-type cells can also be induced to express the tdy1 mutant phenotype. In every tdy1 phenotypic region analyzed, we observed that all five TLs expressed the tdy1 anthocyanin- and starch-accumulating phenotypes, regardless of whether the TLs were genetically mutant or wild type. We never observed instances of mixed phenotypic layers in which one or more TLs expressed a tdy1 mutant phenotype while the others did not. Signifi- cantly, in every chimeric sector analyzed that expressed a tdy1 phenotype, the TL3 was mutant. This suggests that the site of Tdy1 function is within the innermost leaf layer (Fig. 5E). This is supported by the observation that the genotype of the epidermal and subepidermal mesophyll cell layers had no influence on determining the phenotype of the tissue (Figs. 5E, 6, G and H, and 7, D, H, L, and P; Supplemental Table S1). If the Tdy1 gene acts cell autonomously, tdy1 phenotypic regions should form in the albino mutant sectors and extend to the border of green, wild-type tissue. Alternatively, if Tdy1 functions completely non- cell autonomously, no tdy1 regions should occur in the white tissue, as Tdy1 function in neighboring wild- type cells would generate a mobile product that can complement the mutation in albino tissues. As evidenced by the pronounced anthocyanin accumulation, the tdy1 phenotype was expressed in albino tissue (Figs. 5B and 8, A and B; Supplemental Fig. S2). This indicates Tdy1 does not generate a signal that is able to move laterally over large distances. However, the tdy1 phenotypic regions never reached the mosaic sector border. We always observed a narrow strip of albino tissue lacking anthocyanin positioned between the wild-type and mutant anthocyanin-accumulating region. Cross sections through albino sectors displaying this compensatory effect show decreasing anthocyanin as the tdy1 region approaches the wild-type tissue (Fig. 8C). IKI staining of these sections reveals that starch accumulation also ...
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... and starch, demonstrating that yg revertant tissue expresses all aspects of the tdy1 phenotype (Fig. 4, E, F, and H). Yg - str* pale-green mutant tissue displays reduced chlorophyll abundance (Fig. 4I) yet increased chlorophyll autofluorescence (Fig. 4J) similar to some high-chlorophyll fluorescence mutants (Miles and Daniel, 1974). Yg - str* pale-green mutant tissues do not accumulate starch (Fig. 4K). Occasionally, a tdy1 region spread into adjacent Yg - str* pale- green mutant tissue (Fig. 4, B, arrows, and L), and the chlorophyll autofluorescence seen in pale-green mutant tissue was reduced (compare Fig. 4, J and M). These tissues expressed anthocyanin and exhibited starch accumulation in mesophyll cells similar to tdy1 regions in yg revertant tissue (Fig. 4, H and N). As tdy1 regions only initiate within yg revertant tissue, these data suggest functional chloroplasts are needed to generate the Suc that overaccumulates and induces tdy1 phenotypic expression. Additionally, if sufficient levels accumulate, Suc can move into neighboring pale-green mutant cells, causing them to express the tdy1 phenotype (Fig. 4, B and L–N), as seen in ij1; tdy1 plants. As chloroplasts are needed to generate the sugars that induce tdy1 phenotypic expression, one possibility is that Tdy1 functions in photosynthetic cells. To test this hypothesis and determine the cell autonomy of Tdy1 , we performed a clonal mosaic analysis. Based on the ij1; tdy1 double mutant studies, we knew that tdy1 regions can be detected in albino tissues by anthocyanin and starch accumulation (Fig. 3). Genetic stocks were constructed in which tdy1 was linked in coupling to the proximally located albino mutant white14 ( w14 ), and the homologous chromosome carried wild-type functional alleles of both genes (Fig. 5A; Supplemental Fig. S1). g irradiation of germinating seeds induced chromosome breakage and uncovered albino, aneuploid w14 tdy1 / 2 sectors present in otherwise wild- type green plants. We analyzed white tissues marked by anthocyanin pigmentation to determine which cell layers lacked wild-type Tdy1 function and thereby resulted in expression of the tdy1 phenotype (Fig. 5B). In this experiment, we observed tdy1 regions within albino tissue (Figs. 5B and 8A; Supplemental Fig. S2). This suggests that wild-type cells containing functional chloroplasts produce the Suc that moves into adjoining mutant tissue, in which the absence of TDY1 results in Suc overaccumulation and a mutant phenotype. For describing tissue layers (TLs) in the transverse dimension, we utilized the numbering system of Nelson et al. (2002; Fig. 5D). In brief, adaxial and abaxial epidermal layers are respectively termed TL1 and TL5, subepidermal mesophyll layers TL2 and TL4, and the innermost layer, containing the veins, bundle sheath cells, and interveinal mesophyll cells, is termed TL3. Chimeric sectors analyzed in the mosaic analysis are summarized in Figure 5E and Supplemental Table S1. Ten genotypic classes (i–x) of chimeric sectors expressed a tdy1 phenotype. In examining the distribution of tdy1 regions within mosaic leaves, there was no effect whether the albino sectors were located on lower (juvenile) or upper (adult) leaves, nor were differences found between a single albino sector on an isolated leaf or on a meristematic sector encompassing multiple leaves. Likewise, sector position within the lateral dimension of the leaf had no effect on tdy1 phenotypic expression (Supplemental Table S1). Examination of w14 hemizygous tissue from control plants not carrying tdy1 showed aneuploidy for chromosome 6 did not result in anthocyanin deposition (Fig. 5C) or produce any adverse affects on leaf development, as previously reported (Walker and Smith, 2002). To genetically dissect the site and mode of Tdy1 function, we investigated whether a wild-type epidermis prevented expression of the mutant phenotype. For each albino sector, the epidermal genotype was determined by examining guard cells for chlorophyll autofluorescence, as these are the only epidermal cells containing chloroplasts (Fig. 6, A and B). In wild-type tissue, guard cell chloroplasts can be seen under blue light as two bright red points within each guard cell pair (Fig. 6, B, F, H, and J). Free-hand transverse cross sections of the tissue were inspected to determine the genotype of internal TLs. In entirely albino tissues expressing the tdy1 phenotype (Fig. 6C), chlorophyll autofluorescence is absent from guard cells (Figs. 5E [class i] and 6D). However, we identified multiple sectors in which a genetically wild-type epidermal layer(s) overlaid entirely mutant internal layers and exhibited a tdy1 phenotype, as marked by anthocyanin accumulation (Figs. 5E [classes iv–vi] and 6, E and F). Consistent with this, we observed an epidermal meri- clinal sector where wild-type and mutant tissues are juxtaposed, and both strongly display the tdy1 anthocyanin-accumulating phenotype regardless of their genotype (Fig. 6, G and H). Other periclinal chimeric sectors with a wild-type epidermal layer and different internal layers composed of genetically wild-type or mutant layers also expressed a tdy1 mutant phenotype (Fig. 5E, classes vii–x). For instance, in a class x chimeric sector, despite both epidermal layers and one of the internal layers being wild type, the epidermal layers expressed a tdy1 phenotype (Figs. 5E and 6, I and J). In every case examined, wild-type Tdy1 function in the epidermis was not sufficient to prevent a tdy1 region from forming. Thus, independent of its genotype, the epidermis phenotypically reflected the phenotype of the internal layers. These data also demonstrate that genotypically wild-type Tdy1 epidermal cells can be induced to express the tdy1 mutant phenotype, presumably due to accumulation and movement of Suc from underlying mutant cells. To investigate in which internal TLs Tdy1 functions and to determine its cell autonomy, aneuploid w14 tdy1 / 2 chimeric sectored leaves expressing tdy1 regions were examined. As shown previously, wild-type tissue displays abundant chlorophyll levels and shows starch accumulation only in bundle sheath cells (Fig. 7, A–D). White w14 tdy1 / 2 sectors not expressing anthocyanin lacked both chlorophyll and starch (Fig. 7, E–H). In contrast, w14 tdy1 / 2 albino sectors containing tdy1 phenotypic regions marked by anthocyanin lacked chlorophyll (Fig. 7, I and J) and accumulated starch in both mesophyll and bundle sheath cells (Fig. 7, K and L). These results show aneuploid leaf sectors express the tdy1 phenotype. In periclinal chimeras, tdy1 regions were observed only in instances where the TL3 was mutant (Fig. 5E [classes ii–x]; Supplemental Table S1). This can be seen in an example where part of the TL4 was genetically wild type, as evidenced by the presence of chlorophyll, but exhibited the diminished chlorophyll abundance of a tdy1 region (Fig. 7, M and N; compare with Figs. 2B and 7B). Moreover, the TL4 mesophyll cells accumulated starch, which is a hallmark of tdy1 phenotypic expression (Fig. 7, O and P). Hence, internal wild-type cells can also be induced to express the tdy1 mutant phenotype. In every tdy1 phenotypic region analyzed, we observed that all five TLs expressed the tdy1 anthocyanin- and starch-accumulating phenotypes, regardless of whether the TLs were genetically mutant or wild type. We never observed instances of mixed phenotypic layers in which one or more TLs expressed a tdy1 mutant phenotype while the others did not. Signifi- cantly, in every chimeric sector analyzed that expressed a tdy1 phenotype, the TL3 was mutant. This suggests that the site of Tdy1 function is within the innermost leaf layer (Fig. 5E). This is supported by the observation that the genotype of the epidermal and subepidermal mesophyll cell layers had no influence on determining the phenotype of the tissue (Figs. 5E, 6, G and H, and 7, D, H, L, and P; Supplemental Table S1). If the Tdy1 gene acts cell autonomously, tdy1 phenotypic regions should form in the albino mutant sectors and extend to the border of green, wild-type tissue. Alternatively, if Tdy1 functions completely non- cell autonomously, no tdy1 regions should occur in the white tissue, as Tdy1 function in neighboring wild- type cells would generate a mobile product that can complement the mutation in albino tissues. As evidenced by the pronounced anthocyanin accumulation, the tdy1 phenotype was expressed in albino tissue (Figs. 5B and 8, A and B; Supplemental Fig. S2). This indicates Tdy1 does not generate a signal that is able to move laterally over large distances. However, the tdy1 phenotypic regions never reached the mosaic sector border. We always observed a narrow strip of albino tissue lacking anthocyanin positioned between the wild-type and mutant anthocyanin-accumulating region. Cross sections through albino sectors displaying this compensatory effect show decreasing anthocyanin as the tdy1 region approaches the wild-type tissue (Fig. 8C). IKI staining of these sections reveals that starch accumulation also diminishes as the tdy1 region nears wild-type tissue (Fig. 8D). Hence, proximity to wild- type tissue has a preventative effect on tdy1 mutant phenotypic expression but only for a limited distance. Therefore, Tdy1 acts non-cell autonomously over a limited distance. tdy1 mutant leaves display chlorotic regions resulting from the hyperaccumulation of carbohydrates. These regions form during a limited period in leaf development as the leaf emerges from the whorl (Braun et al., 2006). Therefore, TDY1 must function at or prior to this stage to prevent excess carbohydrate accumulation. As elaborated below, one possibility for TDY1 function is to promote the activity of veins to transport Suc out of the tissue and lower its concentration. Consistent with this, our results demonstrate that a chloroplast-derived product, likely Suc, induces the tdy1 phenotype and that Tdy1 acts in the TL ...
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... the mutation abrogates chloroplast function (D. Braun, unpublished data). In Yg - str*; tdy1 double mutant leaves, tdy1 phenotypic regions are indicated by the accumulation of anthocyanin. We found that these regions localize to the revertant green yg tissue (Fig. 4B). The only exceptions to this observation occurred if a tdy1 region was sufficiently large (Fig. 4B, arrows). To verify the cellular expression of the tdy1 phenotype, we examined tissues from Yg - str*; tdy1 double mutant leaves for chlorophyll and starch accumulation (Fig. 4, C–N). Green tissue in revertant yg sectors displays greater chlorophyll levels than in tdy1 phenotypic regions (Fig. 4, C, D, F, and G). Additionally, tdy1 regions in revertant yg tissue accumulate anthocyanin and starch, demonstrating that yg revertant tissue expresses all aspects of the tdy1 phenotype (Fig. 4, E, F, and H). Yg - str* pale-green mutant tissue displays reduced chlorophyll abundance (Fig. 4I) yet increased chlorophyll autofluorescence (Fig. 4J) similar to some high-chlorophyll fluorescence mutants (Miles and Daniel, 1974). Yg - str* pale-green mutant tissues do not accumulate starch (Fig. 4K). Occasionally, a tdy1 region spread into adjacent Yg - str* pale- green mutant tissue (Fig. 4, B, arrows, and L), and the chlorophyll autofluorescence seen in pale-green mutant tissue was reduced (compare Fig. 4, J and M). These tissues expressed anthocyanin and exhibited starch accumulation in mesophyll cells similar to tdy1 regions in yg revertant tissue (Fig. 4, H and N). As tdy1 regions only initiate within yg revertant tissue, these data suggest functional chloroplasts are needed to generate the Suc that overaccumulates and induces tdy1 phenotypic expression. Additionally, if sufficient levels accumulate, Suc can move into neighboring pale-green mutant cells, causing them to express the tdy1 phenotype (Fig. 4, B and L–N), as seen in ij1; tdy1 plants. As chloroplasts are needed to generate the sugars that induce tdy1 phenotypic expression, one possibility is that Tdy1 functions in photosynthetic cells. To test this hypothesis and determine the cell autonomy of Tdy1 , we performed a clonal mosaic analysis. Based on the ij1; tdy1 double mutant studies, we knew that tdy1 regions can be detected in albino tissues by anthocyanin and starch accumulation (Fig. 3). Genetic stocks were constructed in which tdy1 was linked in coupling to the proximally located albino mutant white14 ( w14 ), and the homologous chromosome carried wild-type functional alleles of both genes (Fig. 5A; Supplemental Fig. S1). g irradiation of germinating seeds induced chromosome breakage and uncovered albino, aneuploid w14 tdy1 / 2 sectors present in otherwise wild- type green plants. We analyzed white tissues marked by anthocyanin pigmentation to determine which cell layers lacked wild-type Tdy1 function and thereby resulted in expression of the tdy1 phenotype (Fig. 5B). In this experiment, we observed tdy1 regions within albino tissue (Figs. 5B and 8A; Supplemental Fig. S2). This suggests that wild-type cells containing functional chloroplasts produce the Suc that moves into adjoining mutant tissue, in which the absence of TDY1 results in Suc overaccumulation and a mutant phenotype. For describing tissue layers (TLs) in the transverse dimension, we utilized the numbering system of Nelson et al. (2002; Fig. 5D). In brief, adaxial and abaxial epidermal layers are respectively termed TL1 and TL5, subepidermal mesophyll layers TL2 and TL4, and the innermost layer, containing the veins, bundle sheath cells, and interveinal mesophyll cells, is termed TL3. Chimeric sectors analyzed in the mosaic analysis are summarized in Figure 5E and Supplemental Table S1. Ten genotypic classes (i–x) of chimeric sectors expressed a tdy1 phenotype. In examining the distribution of tdy1 regions within mosaic leaves, there was no effect whether the albino sectors were located on lower (juvenile) or upper (adult) leaves, nor were differences found between a single albino sector on an isolated leaf or on a meristematic sector encompassing multiple leaves. Likewise, sector position within the lateral dimension of the leaf had no effect on tdy1 phenotypic expression (Supplemental Table S1). Examination of w14 hemizygous tissue from control plants not carrying tdy1 showed aneuploidy for chromosome 6 did not result in anthocyanin deposition (Fig. 5C) or produce any adverse affects on leaf development, as previously reported (Walker and Smith, 2002). To genetically dissect the site and mode of Tdy1 function, we investigated whether a wild-type epidermis prevented expression of the mutant phenotype. For each albino sector, the epidermal genotype was determined by examining guard cells for chlorophyll autofluorescence, as these are the only epidermal cells containing chloroplasts (Fig. 6, A and B). In wild-type tissue, guard cell chloroplasts can be seen under blue light as two bright red points within each guard cell pair (Fig. 6, B, F, H, and J). Free-hand transverse cross sections of the tissue were inspected to determine the genotype of internal TLs. In entirely albino tissues expressing the tdy1 phenotype (Fig. 6C), chlorophyll autofluorescence is absent from guard cells (Figs. 5E [class i] and 6D). However, we identified multiple sectors in which a genetically wild-type epidermal layer(s) overlaid entirely mutant internal layers and exhibited a tdy1 phenotype, as marked by anthocyanin accumulation (Figs. 5E [classes iv–vi] and 6, E and F). Consistent with this, we observed an epidermal meri- clinal sector where wild-type and mutant tissues are juxtaposed, and both strongly display the tdy1 anthocyanin-accumulating phenotype regardless of their genotype (Fig. 6, G and H). Other periclinal chimeric sectors with a wild-type epidermal layer and different internal layers composed of genetically wild-type or mutant layers also expressed a tdy1 mutant phenotype (Fig. 5E, classes vii–x). For instance, in a class x chimeric sector, despite both epidermal layers and one of the internal layers being wild type, the epidermal layers expressed a tdy1 phenotype (Figs. 5E and 6, I and J). In every case examined, wild-type Tdy1 function in the epidermis was not sufficient to prevent a tdy1 region from forming. Thus, independent of its genotype, the epidermis phenotypically reflected the phenotype of the internal layers. These data also demonstrate that genotypically wild-type Tdy1 epidermal cells can be induced to express the tdy1 mutant phenotype, presumably due to accumulation and movement of Suc from underlying mutant cells. To investigate in which internal TLs Tdy1 functions and to determine its cell autonomy, aneuploid w14 tdy1 / 2 chimeric sectored leaves expressing tdy1 regions were examined. As shown previously, wild-type tissue displays abundant chlorophyll levels and shows starch accumulation only in bundle sheath cells (Fig. 7, A–D). White w14 tdy1 / 2 sectors not expressing anthocyanin lacked both chlorophyll and starch (Fig. 7, E–H). In contrast, w14 tdy1 / 2 albino sectors containing tdy1 phenotypic regions marked by anthocyanin lacked chlorophyll (Fig. 7, I and J) and accumulated starch in both mesophyll and bundle sheath cells (Fig. 7, K and L). These results show aneuploid leaf sectors express the tdy1 phenotype. In periclinal chimeras, tdy1 regions were observed only in instances where the TL3 was mutant (Fig. 5E [classes ii–x]; Supplemental Table S1). This can be seen in an example where part of the TL4 was genetically wild type, as evidenced by the presence of chlorophyll, but exhibited the diminished chlorophyll abundance of a tdy1 region (Fig. 7, M and N; compare with Figs. 2B and 7B). Moreover, the TL4 mesophyll cells accumulated starch, which is a hallmark of tdy1 phenotypic expression (Fig. 7, O and P). Hence, internal wild-type cells can also be induced to express the tdy1 mutant phenotype. In every tdy1 phenotypic region analyzed, we observed that all five TLs expressed the tdy1 anthocyanin- and starch-accumulating phenotypes, regardless of whether the TLs were genetically mutant or wild type. We never observed instances of mixed phenotypic layers in which one or more TLs expressed a tdy1 mutant phenotype while the others did not. Signifi- cantly, in every chimeric sector analyzed that expressed a tdy1 phenotype, the TL3 was mutant. This suggests that the site of Tdy1 function is within the innermost leaf layer (Fig. 5E). This is supported by the observation that the genotype of the epidermal and subepidermal mesophyll cell layers had no influence on determining the phenotype of the tissue (Figs. 5E, 6, G and H, and 7, D, H, L, and P; Supplemental Table S1). If the Tdy1 gene acts cell autonomously, tdy1 phenotypic regions should form in the albino mutant sectors and extend to the border of green, wild-type tissue. Alternatively, if Tdy1 functions completely non- cell autonomously, no tdy1 regions should occur in the white tissue, as Tdy1 function in neighboring wild- type cells would generate a mobile product that can complement the mutation in albino tissues. As evidenced by the pronounced anthocyanin accumulation, the tdy1 phenotype was expressed in albino tissue (Figs. 5B and 8, A and B; Supplemental Fig. S2). This indicates Tdy1 does not generate a signal that is able to move laterally over large distances. However, the tdy1 phenotypic regions never reached the mosaic sector border. We always observed a narrow strip of albino tissue lacking anthocyanin positioned between the wild-type and mutant anthocyanin-accumulating region. Cross sections through albino sectors displaying this compensatory effect show decreasing anthocyanin as the tdy1 region approaches the wild-type tissue (Fig. 8C). IKI staining of these sections reveals that starch accumulation also diminishes as the tdy1 region nears wild-type tissue (Fig. 8D). Hence, proximity to wild- type tissue has a preventative effect on tdy1 mutant ...
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... Previous studies from our and other groups have shown that lack of grain sink resulting from unfertilized maize (Zea mays L.) ears results in increased sugar accumulation in leaves and premature senescence (Christensen et al. 1981;Ceppi et al. 1987;Sekhon et al. 2012;Kumar et al. 2019). The inefficient partitioning of sugars observed in sugar transporter mutants often produces a senescence-like phenotype in maize (Baker and Braun 2007;Slewinski et al. 2012). Likewise, the application of sucrose (Suc) to leaf in Arabidopsis (Arabidopsis thaliana) and hyperaccumulation of sugars in barley (Hordeum vulgare) leaves due to stem girdling resulted in premature senescence (Parrott et al. 2007;Wingler et al. 2009). ...
Source and sink interactions play a critical but mechanistically poorly understood role in the regulation of senescence. To disentangle the genetic and molecular mechanisms underlying source-sink regulated senescence (SSRS), we performed a phenotypic, transcriptomic, and systems genetics analysis of senescence induced by the lack of a strong sink in maize (Zea mays). Comparative analysis of genotypes with contrasting SSRS phenotypes revealed that feedback inhibition of photosynthesis, a surge in reactive oxygen species, and the resulting endoplasmic reticulum (ER) stress were the earliest outcomes of weakened sink demand. Multi-environmental evaluation of a biparental population and a diversity panel identified 12 quantitative trait loci and 24 candidate genes, respectively, underlying SSRS. Combining the natural diversity and coexpression networks analyses identified seven high-confidence candidate genes involved in proteolysis, photosynthesis, stress response, and protein folding. The role of a cathepsin B like protease 4 (ccp4), a candidate gene supported by systems genetic analysis, was validated by analysis of natural alleles in maize and heterologous analyses in Arabidopsis (Arabidopsis thaliana). Analysis of natural alleles suggested that a 700-bp polymorphic promoter region harboring multiple ABA-responsive elements is responsible for differential transcriptional regulation of ccp4 by ABA and the resulting variation in SSRS phenotype. We propose a model for SSRS wherein feedback inhibition of photosynthesis, ABA signaling, and oxidative stress converge to induce ER stress manifested as programmed cell death and senescence. These findings provide a deeper understanding of signals emerging from loss of sink strength and offer opportunities to modify these signals to alter senescence program and enhance crop productivity.
... There is also evidence that insufficient export of starch degradation products can cause chlorosis and chloroplast destruction in Arabidopsis [60]. Similarly, the tie-dyed1 maize mutant unable to export carbohydrates as good as wild-type plants exhibited strong light-dependent chlorosis, presumably due to the carbohydrate accumulation [61][62][63]. Accordingly, a high demand for assimilates from sink tissues was shown to reduce the damage. When two onion species were grown under CL, the bulb-forming species Allium cepa L. showed no photosynthesis inhibition, whereas photosynthesis of a non-bulbing onion A. fistulosum L. became depressed [59]. ...
... The accumulation of carbohydrates under CL can cause, among other things, disorders and injuries in plants due to enhanced generation of reactive oxygen species (ROS) [5]. The accumulation of carbohydrates is associated with the suppression of photosynthesis under diverse conditions, including long-term exposure to high CO 2 concentrations [74], magnesium deficiency [75], feeding leaves with sugars [76], and introducing mutations that affect carbohydrate metabolism [62,63]. Photosynthetic regulation, including photosynthesis inhibition and retrograde signaling, is a series of short-term and long-term adaptive changes aimed at adjusting the rates of ATP and NADPH production in the light reactions to keep RUSSIAN them in balance with the consumption rates of these products to prevent the overreduction of chloroplast ETC components [77]. ...
... Mg fertilizer has been reported for considerably increasing the yield and quality of wax gourds as per the data taken from the five-year trial (Table 1) [20,29]. Yield formation acquires sugars which are largely generated via photosynthetic assimilation in leaves and are further metabolized and sink into phloem, thus promoting fruit growth [37][38][39][40][41]. Interruptions on carbon assimilates in leaves and/or the transport capacity of phloem have been widely reported to be associated with reductions in yield [42][43][44][45]. Interestingly, lower carbohydrates in source leaves have been observed both in Mg deficient [46,47] and sufficient conditions [8,11,12,48] but with a different mechanism and consequence. Briefly, the leaf sugar reduces slightly under Mg deficiency, mainly through a restricted photosynthetic capacity in the leaf and is typically identified at an early stage [49,50]. ...
Magnesium (Mg) is critical for agricultural production and human health. The wax gourd yield was greatly affected by Mg fertilizer and it serves as an excellent crop to study Mg functioning in sink growth; however, as a Cucurbitaceae plant which is called a raffinose family oligosaccharides (RFOs)-transporting plant, its adaptive mechanism of photoassimilates transportation and distribution to Mg nutrients remains unknown. Herein, we used two Mg treatments (+Mg 90 kg ha−1; CK as control 0 kg ha−1) to observe the effects of Mg on the photoassimilates status in the leaves, phloem sap and fruit of wax gourd grown across the entire growth stage under a field condition. For the first time, we confirmed that stachyose and raffinose, which accounted for 54.9–78.0% of the total carbohydrates across the whole growth phase in the phloem sap, were the most predominant sugars used for the long-distance transport of wax gourds. They were strongly increased by Mg application which started from the seedling stage to the end of growing season. This response was earlier and more dramatic than the over-accumulation of sucrose in leaves regardless of positions. Hexoses (glucose plus fructose) were the main soluble sugars in the source leaves as well as in the mesocarp tissues, and their responses to Mg varied with temporal and spatial differences. The difference in the sugar status in the leaves between the upper, middle and lower positions is closely related to the concentration of Mg ions. Compared with Mg deficiency (CK), Mg application stimulated sugar accumulation in the leaves at the seedling and flowering stages by 10.6–24.5%. On the contrary, after fruit set, Mg supplementation significantly reduced the soluble sugar concentration in leaves by 33.5–67.9%. Mg treatment also spiked the fructose and glucose in fruit at the fruit expansion stage by 26.7% and 16.4%, respectively. Taken together, our results showed that Mg tailors the carbohydrate status in the source (leaves), flow (phloem sap) and sink (mesocarp tissues) in wax gourds, especially during the fruit growth period. The lower stachyose in the phloem at the beginning of fruit setting may be an early indication of a curtailed sink process in wax gourds grown in Mg deficient soil.
... The metabolic pathways of storage starch and transitory starch are different. There are many studies focusing on the metabolism of storage starch in various plant species, but few studies have been performed on the regulation of transitory starch metabolism in plant leaves, except those in Arabidopsis (Zhang et al. 2005;Kötting et al. 2009;Tsai et al. 2009), maize (Baker and Braun 2007;Slewinski et al. 2009), and rice (Asatsuma et al. 2005;Huang et al. 2020). The metabolism of leaf starch is of central importance for the balance of sink and source in plants and affects plant growth and development (Schulze et al. 1991;Corbesier et al. 1998;Paul and Foyer 2001). ...
Starch is an important primary metabolite in plants, which can provide bioenergy for fuel ethanol production. There are many studies focusing on starch metabolism in Arabidopsis, maize, and rice, but few reports have been made on the starch content of tobacco leaves. Hence, to identify the marker-trait associations and isolate the candidate genes related to starch content of tobacco leaf, the genome-wide association study (GWAS) was performed using a multiparent advanced generation intercross (MAGIC) population consisting of 276 accessions genotyped by a 430 K SNP array. In this study, we detected the leaf starch content of tobacco plants cultivated in two places (Zhucheng and Chenzhou), which showed a wide variation of starch content in the population. A total of 28 and 45 significant single-nucleotide polymorphism (SNP) loci associated with leaf starch content were identified by single-locus and multi-locus GWAS models, respectively, and the phenotypic variance explained by these loci varied from 1.80 to − 14.73%. Furthermore, among these quantitative trait loci (QTLs), one SNP, AX-106011713 located on chromosome 19, was detected repeatedly in multiple models and two environments, which was selected for linkage disequilibrium (LD) analysis to obtain the target candidate region. Through gene annotation, haplotype, and gene expression analysis, two candidate genes encoding E3 ubiquitin-protein ligase (Ntab0823160) and fructose-bisphosphate aldolase (Ntab0375050) were obtained. Results showed that the variety carrying the beneficial alleles of the two candidate genes had higher gene expression level and leaf starch content, suggesting the potential role of candidate genes in enhancing the level of tobacco leaf starch content. Furthermore, silencing of Ntab0823160 in tobacco leaves reduced the content of total starch to 39.41–69.75% of that in the wide type plants. Taken together, our results provide useful resources for further investigation of the starch metabolic pathway and are also beneficial for the creation of eco-friendly cultivars with increased accumulation of leaf starch content.
... Leaves were cleared and stained with IKI as previously described (Baker and Braun, 2007). ...
Carbohydrate partitioning from leaves to sink tissues is essential for plant growth and development. The maize (Zea mays) recessive carbohydrate partitioning defective28 (cpd28) and cpd47 mutants exhibit leaf chlorosis and accumulation of starch and soluble sugars. Transport studies with 14C-sucrose (Suc) found drastically decreased export from mature leaves in cpd28 and cpd47 mutants relative to wild-type siblings. Consistent with decreased Suc export, cpd28 mutants exhibited decreased phloem pressure in mature leaves, and altered phloem cell wall ultrastructure in immature and mature leaves. We identified the causative mutations in the Brittle Stalk2-Like3 (Bk2L3) gene, a member of the COBRA family, which is involved in cell wall development across angiosperms. None of the previously characterized COBRA genes are reported to affect carbohydrate export. Consistent with other characterized COBRA members, the BK2L3 protein localized to the plasma membrane, and the mutants condition a dwarf phenotype in dark-grown shoots and primary roots, as well as the loss of anisotropic cell elongation in the root elongation zone. Likewise, both mutants exhibit a significant cellulose deficiency in mature leaves. Therefore, Bk2L3 functions in tissue growth and cell wall development, and this work elucidates a unique connection between cellulose deposition in the phloem and whole-plant carbohydrate partitioning.
... Studies have been conducted on anthocyanins accumulation, considering nutritional stress by limiting the amounts of nutrients in the medium (Schiozer and Barata, 2007). Deficiency of nutrients especially nitrogen, phosphorus and sulfur, are accompanied by anthocyanin accumulation in plants as a strategy to avoid the over-accumulation of carbohydrates in tissues and to prevent physiological disorders (Baker and Braun, 2007). Apart from the overall concentration of total nitrogen, the ratio of ammonium (NH 4 + ) to nitrate (NO 3 -) has also been shown to markedly affect the production of anthocyanin. ...
Anthocyanins, the color compounds of plants, are known for their wide applications in food, nutraceuticals and cosmetic industry. The biosynthetic pathway of anthocyanins is well established with the identification of potential key regulatory genes, which make it possible to modulated its production by biotechnological means. Various biotechnological systems, including use of in vitro plant cell or tissue cultures as well as microorganisms have been used for the production of anthocyanins under controlled conditions, however, a wide range of factors affects their production. In addition, metabolic engineering technologies have also used the heterologous production of anthocyanins in recombinant plants and microorganism. However, these approaches have mostly been tested at the lab- and pilot-scales, while very few up-scaling studies have been undertaken. Various challenges and ways of investigation are proposed here to improve anthocyanin production by using the in vitro plant cell or tissue culture and metabolic engineering of plants and microbial culture systems. All these methods are capable of modulating the producing anthocyanins, which can be further utilized for pharmaceutical, cosmetics and food applications.
... Identification of two sugar transporters (ZmSWEET1b and ZmMST), a cell wall invertase (incw4), and a mitochondrial pentatricopeptide repeat protein (dek10) further supports a key role for sugar partitioning in senescence. Disruption of Suc transport from leaves often leads to senescence-like symptoms in maize as seen in sugar transport mutants tdy1, tdy2, and sut1 (Baker and Braun, 2007;Slewinski et al., 2009Slewinski et al., , 2012. Arabidopsis hexose transporter STP13, a putative ortholog of ZmMST4, is associated with senescence and nitrogen use efficiency (Nørholm et al., 2006;Schofield et al., 2009), and putative rice ortholog, OsMST4, transports hexoses generated by cell wall invertases (Wang et al., 2007). ...
Premature senescence in annual crops reduces yield, while delayed senescence, termed stay-green, imposes positive and negative impacts on yield and nutrition quality. Despite its importance, scant information is available on the genetic architecture of senescence in maize (Zea mays) and other cereals. We combined a systematic characterization of natural diversity for senescence in maize and coexpression networks derived from transcriptome analysis of normally senescing and stay-green lines. Sixty-four candidate genes were identified by genome-wide association study (GWAS), and 14 of these genes are supported by additional evidence for involvement in senescence-related processes including proteolysis, sugar transport and signaling, and sink activity. Eight of the GWAS candidates, independently supported by a coexpression network underlying stay-green, include a trehalose-6-phosphate synthase, a NAC transcription factor, and two xylan biosynthetic enzymes. Source-sink communication and the activity of cell walls as a secondary sink emerge as key determinants of stay-green. Mutant analysis supports the role of a candidate encoding Cys protease in stay-green in Arabidopsis (Arabidopsis thaliana), and analysis of natural alleles suggests a similar role in maize. This study provides a foundation for enhanced understanding and manipulation of senescence for increasing carbon yield, nutritional quality, and stress tolerance of maize and other cereals.
... SWEETs mediate transport of Suc from the site of synthesis to apoplasm (Chen et al., 2012;Eom et al., 2015), ZmSUT2 facilitates transient storage of Suc in the vacuole during the day time (Leach, Tran, Slewinski, Meeley, & Braun, 2017), and ZmSUT1 facilitates phloem loading by importing of Suc from apoplast to CC/SE complex (Slewinski, Garg, Johal, & Braun, 2010;Slewinski, Meeley, & Braun, 2009). Hyperaccumulation of sugars in mutants lacking sugar transporters results in the appearance of senescence-like symptoms in maize (Baker & Braun, 2007;Slewinski, Baker, Stubert, & Braun, 2012). However, only ZmSUT2 showed a clear association with sugar partitioning and SSRS owing to high transcript levels of this transporter in PHG35 albeit late in the development (33 and 36 DAA). ...
Source‐sink communication is one of the key regulators of senescence; however, the mechanisms underlying such regulation are largely unknown. We analyzed senescence induced by the lack of grain sink in maize, termed source‐sink regulated senescence (SSRS), and compared the associated physiological and metabolic changes with those accompanying natural senescence. Phenotypic characterization 31 diverse field‐grown inbreds revealed substantial variation for both SSRS and natural senescence. Partitioning of excess carbohydrates to alternative sinks, mainly internodes and husks, emerged as a critical mechanism underlying both SSRS and stay‐green. Time‐course analyses of SSRS sensitive (B73) and resistant (PHG35) inbreds confirmed the role of sugar partitioning in SSRS and stay‐green. Elevated hemicellulose content in PHG35 internodes highlighted the role of the cell wall as a significant alternative sink. Sugar signaling emerged as an important regulator of SSRS as evident from an increased accumulation of trehalose‐6‐phosphate and decreased transcript levels of snf1‐related protein kinase1, two signaling components associated with senescence, in B73. These findings demonstrate a crucial role of sugar partitioning, signaling, and utilization in SSRS. Available genetic variation for SSRS and a better understanding of the underlying mechanisms would help modify sugar partitioning and senescence to enhance the productivity of maize and related grasses.
... Starch staining was performed as previously described (Ruzin, 1999). For maize leaves, the cpd33 mutant and wild-type leaves were collected at 5 AM, boiled with 95% ethanol to clear the photosynthetic pigments, and stained with 1% IKI (Baker and Braun, 2007). Arabidopsis leaves were harvested at the end of the day, at 2 hours into the night period, or at the end of the night, cleared with 95% ethanol, and stained with 1% IKI. ...
To sustain plant growth, development, and crop yield, sucrose must be transported from leaves to distant parts of the plant, such as seeds and roots. To identify genes that regulate sucrose accumulation and transport in maize (Zea mays), we isolated carbohydrate partitioning defective33 (cpd33), a recessive mutant that accumulated excess starch and soluble sugars in mature leaves. The cpd33 mutants also exhibited chlorosis in the leaf blades, greatly diminished plant growth, and reduced fertility. Cpd33 encodes a protein containing multiple C2 domains and transmembrane regions. Subcellular localization experiments showed the CPD33 protein localized to plasmodesmata (PD), the plasma membrane, and the endoplasmic reticulum. We also found that a loss-of-function mutant of the CPD33 homolog in Arabidopsis, QUIRKY, had a similar carbohydrate hyperaccumulation phenotype. Radioactively labeled sucrose transport assays showed that sucrose export was significantly lower in cpd33 mutant leaves relative to wild-type leaves. However, PD transport in the adaxial-abaxial direction was unaffected in cpd33 mutant leaves. Intriguingly, transmission electron microscopy revealed fewer PD at the companion cell-sieve element interface in mutant phloem tissue, providing a possible explanation for the reduced sucrose export in mutant leaves. Collectively, our results suggest that CPD33 functions to promote symplastic transport into sieve elements.
... Leaves were cleared and stained with iodine-potassium iodide as previously described (Baker and Braun, 2007). ...
Plants synthesize carbohydrates in photosynthetic tissues, with the majority of plants transporting sucrose to non-photosynthetic tissues to sustain growth and development. While the anatomical, biochemical, and physiological processes regulating sucrose long-distance transport are well characterized, little is known concerning the genes controlling whole-plant carbohydrate partitioning. To identify loci influencing carbon export from leaves, we screened mutagenized maize plants for phenotypes associated with reduced carbohydrate transport, including chlorosis and excessive starch and soluble sugars in leaves. Carbohydrate partitioning defective1 (Cpd1) was identified as a semi-dominant mutant exhibiting these phenotypes. Phloem transport experiments suggested that the hyperaccumulation of starch and soluble sugars in the Cpd1/+ mutant leaves was due to inhibited sucrose export. Interestingly, ectopic callose deposits were observed in the phloem of mutant leaves, and likely underlie the decreased transport. In addition to the carbohydrate hyperaccumulation phenotype, Cpd1/+ mutants overaccumulate benzoxazinoid defense compounds and exhibit increased tolerance when attacked by aphids. However, double mutant studies between Cpd1/+ and benzoxazinoid-less plants indicate that the ectopic callose and carbon hyperaccumulation are independent of benzoxazinoid production. Based on the formation of callose occlusions in the developing phloem, we hypothesize that the cpd1 gene functions early in phloem development, thereby impacting whole-plant carbohydrate partitioning.
