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First published online April 13, 2007; 10.1104/pp.107.098814 Plant Physiology 144:867-878 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
tie-dyed1 Functions Non-Cell Autonomously to Control Carbohydrate Accumulation in Maize Leaves1,[C],[W],[OA]Department of Biology, Pennsylvania State University, University Park, Pennsylvania 16802
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 frequently occur at lateral veins. This suggests that lateral veins act to limit the expansion of tdy1 phenotypic regions by transporting Suc out of the tissue. Double mutant studies between tdy1 and chloroplast-impaired mutants demonstrate that functional chloroplasts are needed to generate the Suc that induces the tdy1 phenotype. However, we also found that albino cells can express the tdy1 phenotype and overaccumulate Suc imported from neighboring green tissues. To characterize the site and mode of action of Tdy1, we performed a clonal mosaic analysis. In the transverse dimension, we localized the function of Tdy1 to the innermost leaf layer. Additionally, we determined that if this layer lacks Tdy1, Suc can accumulate, move into adjacent genetically wild-type layers, and induce tdy1 phenotypic expression. In the lateral dimension, we observed that a tdy1 phenotypic region did not reach the mosaic sector boundary, suggesting that wild-type Tdy1 acts non-cell autonomously and exerts a short-range compensatory effect on neighboring mutant tissue. A model proposing that Tdy1 functions in the vasculature to sense high concentrations of sugar, up-regulate Suc transport into veins, and promote tissue differentiation and function is discussed.
As well as being the primary products of carbon assimilation, sugars have been shown to act as signals that regulate plant growth and development, influencing embryogenesis, seed germination, seedling growth, organ initiation, flowering, and senescence (Koch, 2004  ; Gibson, 2005; Raines and Paul, 2006; Rolland et al., 2006). In addition to their role in regulating plant growth, sugars play important roles in controlling gene expression. Numerous studies in a wide variety of plants have found evidence for sugars both inducing and repressing gene expression (Koch, 1996; Chiou and Bush, 1998; Aoki et al., 1999; Fujiki et al., 2001; Lloyd and Zakhleniuk, 2004; Blasing et al., 2005; Crevillen et al., 2005). Perhaps the best known examples of this are high sugar levels repressing photosynthetic gene expression (Sheen, 1990, 1994; Jang and Sheen, 1994; Moore et al., 2003). This can be seen in cases where transport of sugars out of photosynthetic tissues is impaired, and the excess carbohydrates down-regulate photosynthesis and photosynthetic gene expression, thereby resulting in chlorosis (Goldschmidt and Huber, 1992; Riesmeier et al., 1994; Krapp and Stitt, 1995; Burkle et al., 1998; Gottwald et al., 2000; Jeannette et al., 2000).
Maize (Zea mays) leaf blades use C4 carbon assimilation to synthesize carbohydrates (Langdale and Nelson, 1991
Except near the leaf margins, clonal cell lineages within maize leaves are arranged in parallel with the long axis of growth (Poethig and Szymkowiak, 1995
Several features of the tdy1 phenotype suggest that the accumulation and spread of a sugar determines the tissue phenotype. First, within a clonal lineage, cells display different tissue phenotypes (either chlorotic or green), excluding a lineage-dependent mechanism to explain their formation (Fig. 1, A and B). Second, within a tdy1 phenotypic region, all cells display uniform pigmentation, in contrast to what might be predicted if the phenotype of each cell was determined independently. Finally, we observe chlorotic regions surrounded by green tissues, which develop at the same time in a similar environment. This suggests that the external environment alone does not determine the tissue phenotype. Based on these and other observations, we postulated that Tdy1 integrates developmental and physiological information to induce transport of Suc into the veins, thereby preventing the formation of carbohydrate-hyperaccumulating regions in leaves (Braun et al., 2006 ).
We previously found that the first detectable sign of a tdy1 phenotypic region is the hyperaccumulation of starch. This indicates that chlorosis is a secondary effect of excess carbohydrate accumulation and suggests carbohydrates play a role in generating the tdy1 phenotype (Braun et al., 2006
To further investigate where and how Tdy1 functions, we performed a clonal mosaic analysis. We constructed genetic stocks linking an albino mutation to tdy1 as a means to distinguish mutant from wild-type tissue. Radiation was used to induce somatic chromosome breaks and the mosaic sectors analyzed for tdy1 phenotypic expression. If a gene product is able to influence the phenotype of cells outside those in which it is expressed, the gene is considered to act non-cell autonomously. Conversely, if a gene functions only in the cell in which it is produced, it acts cell autonomously (Hake and Sinha, 1994
tdy1 Region Boundaries Are Often Delineated by Lateral Veins
tdy1 leaves exhibit a nonclonal pattern of chlorotic regions that accumulate carbohydrates (Fig. 1B; Braun et al., 2006
Examination of transverse leaf sections from tdy1 region boundaries revealed that lateral veins, the largest vein class in the maize leaf, frequently separated chlorotic and green tissues (Fig. 2
; Table I
). As previously shown, chlorophyll levels in the tdy1 regions were greatly diminished relative to those in green tissue (Fig. 2A). UV illumination verified that the tdy1 regions contain reduced chlorophyll autofluorescence and confirmed that the lateral vein was the border (Fig. 2B). As tdy1 regions hyperaccumulate starch (Braun et al., 2006
Cells Lacking Functional Chloroplasts Can Express the tdy1 Phenotype
The formation of a tdy1 region requires high light, potentially for the production of high levels of Suc (Braun et al., 2006
Histological sections were used to characterize the cellular expression of the tdy1 phenotype in albino tissues. Relative to green tissue (Fig. 3, D and E), tdy1 regions display reduced chlorophyll levels (Fig. 3F) and accumulate excess starch in both bundle sheath and mesophyll cells (Fig. 3G). In ij1 albino tissue, starch was never observed within any cells (Fig. 3, H and I), consistent with albino leaf tissue not accumulating starch (Cox and Dickinson, 1971 ). In ij1; tdy1 albino tissue expressing anthocyanin, starch accumulation was seen in mesophyll and bundle sheath cells, albeit reduced compared to a tdy1 region (Fig. 3, G, J, and K). Because albino tissue can accumulate starch and anthocyanin, it demonstrates that functional chloroplasts are not required for a cell to express the tdy1 phenotype.
In ij1; tdy1 double mutant plants, we did not observe tdy1 regions solely in white tissue. Albino tissues expressing the tdy1 phenotype were always found next to tdy1 regions in green tissues, suggesting functional chloroplasts are necessary to generate the Suc that overaccumulates and induces tdy1 phenotypic expression. To test this hypothesis, double mutants were constructed between tdy1 and Yg-str*. Yg-str* is a dominant, transposon-induced pale-green mutant with revertant sectors of wild-type (yg) dark-green tissue due to somatic excision of the Mutator transposable element (Fig. 4A ). In the absence of Mutator activity, Yg-str* mutants are pale-green seedling lethals, 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).
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 mericlinal 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/– 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/– sectors not expressing anthocyanin lacked both chlorophyll and starch (Fig. 7, E–H). In contrast, w14 tdy1/– 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. Significantly, 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 containing the veins.
At a tdy1 phenotypic region boundary, cells on one side of the lateral vein exhibit the features of a tdy1 chlorotic region, and those on the other side display the characteristics of normal-appearing green tissue. This organization may reflect the half-vein model for maize leaf vascular ontogeny (Langdale et al., 1989
If lateral veins can act as region boundaries, how do tdy1 regions encompass multiple lateral veins? We previously proposed that a threshold level of Suc determines whether the tdy1 phenotype is expressed (Braun et al., 2006
If Suc or another sugar induces the mutant phenotype, we expect functional chloroplasts would be required for its production. To test this hypothesis, we constructed double mutants between tdy1 and Yg-str*. In Yg-str*; tdy1 double mutants, tdy1 phenotypic regions initiated in revertant yg tissue. tdy1 regions did not initiate in pale-green mutant tissue, despite the fact it occupied a greater percentage of leaf area than green revertant tissue. This restriction of tdy1 phenotypic expression demonstrates that functional chloroplasts are needed to generate the Suc required for inducing the tdy1 phenotype. Occasionally, a tdy1 region was large enough that the Suc could spread into adjacent pale-green mutant tissue. In these cases, the fact that pale-green mutant tissue was capable of accumulating starch and anthocyanin indicates functional chloroplasts are not required to express the tdy1 mutant phenotype. This is consistent with our findings from ij1; tdy1 double mutants and albino mosaic sectors. These data support the hypothesis that the inducing substance is a chloroplast by-product, likely Suc.
To understand how Tdy1 functions and to determine its focus of action, we performed a mosaic analysis. In the transverse dimension, we found that Tdy1 function within the epidermal or subepidermal mesophyll layers was not sufficient to prevent tdy1 phenotypic expression. In the absence of Tdy1 function in the TL3, Suc accumulates and moves into all TLs, establishing a tdy1 phenotype throughout the tissue. Therefore, TDY1 functions to limit the accumulation of Suc. Further, the data indicate that Tdy1 acts within the innermost leaf layer. Though it was not possible in this experiment to distinguish where in the TL3 Tdy1 acts, we speculate that Tdy1 functions in the vein. In support of this, we recently cloned Tdy1 and found it encodes a novel protein (Y. Ma and D. Braun, unpublished data). Preliminary expression studies detect the Tdy1 transcript only in veins, confirming the mosaic analysis results. We conclude that Tdy1 functions in the TL3 to limit the accumulation of Suc.
In the lateral dimension, tdy1 phenotypic regions never reached the mosaic sector border, indicating that wild-type tissue produces a non-cell-autonomous compensatory effect over the distance of several veins. That wild-type tissue is apparently both the source of the Suc that induces tdy1 phenotypic expression and produces a short-range compensatory effect suggests these are separate processes. We found that functional chloroplasts are needed to produce sugars that overaccumulate and induce the mutant phenotype. We suggest that the function of vascular tissues, specifically phloem loading of Suc, is responsible for the compensatory effect. Two alternatives could account for the ability of wild-type tissue to prevent neighboring mutant tissue from expressing a tdy1 phenotype (Fig. 9 ). The first possibility is that wild-type tissue generates a short-range mobile signal (either the TDY1 gene product or a downstream transmissible signal) that moves into adjacent mutant tissue and insulates it from the overaccumulation of Suc (Fig. 9A). This would prevent expression of the tdy1 mutant phenotype in the neighboring mutant veins, possibly by promoting their ability to load and transport Suc. Suc transport out of the tissue through the veins would reduce its concentration in the albino tissue adjacent to the mosaic sector border and prevent these tissues from expressing the tdy1 phenotype. The second possibility is that wild-type tissue indirectly affects nearby tdy1 phenotypic expression. For example, veins located in the bordering wild-type tissue might siphon the Suc from neighboring mutant tissue (Fig. 9B). This would lower its concentration below the level needed to induce a tdy1 phenotypic region in the albino tissue located near the mosaic sector border. In either case, the strength with which wild-type tissue could insulate or reduce the Suc concentration in mutant tissue would extend only for a limited distance.
We previously proposed that TDY1 functions as a sugar or osmotic stress sensor that up-regulates an inducible export pathway, resulting in removal of excess sugar from a region of tissue and leading to its normal development (Braun et al., 2006 ). Data presented in this article that lateral veins are frequently associated with tdy1 region boundaries, that chloroplasts are needed to generate the sugar that induces tdy1 phenotypic expression, and that Tdy1 functions in the TL containing the veins support this model. To incorporate the limited non-cell-autonomous action of Tdy1 into the model, we favor the second possibility that TDY1 rescues neighboring mutant tissue indirectly by depleting excess Suc from it. For instance, TDY1 function in wild-type veins may sense high Suc levels and induce SUTs to load Suc into the phloem. The resulting high export capacity in wild-type veins could drain excess Suc from adjacent mutant tissue, lowering Suc concentrations below the threshold necessary to induce a tdy1 region and resulting in normal differentiation. The strength with which wild-type veins influence carbohydrate accumulation in adjacent mutant tissue may vary depending on local Suc concentrations in the tissue. This could account for the variability seen in the size and shape of the compensatory effect. Studies of the maize SUT ZmSUT1 are in agreement with our model and have shown this gene is up-regulated by Suc (Aoki et al., 1999). ZmSUT1 mRNA levels increase in leaves during the day as photosynthates accumulate and decrease steadily during the night as sugar concentrations drop. Moreover, feeding Suc to detached leaves induced ZmSUT1 mRNA levels. To incorporate these data into our model, one possibility is that in developing tdy1 leaves, lack of TDY1 function results in failure to sense high Suc levels and to up-regulate ZmSUT1 or other SUTs in phloem cells, leading to the build up of carbohydrates and the expression of the tdy1 phenotype. Consistent with this model, it was found that antisense expression of NtSUT1 in tobacco (Nicotiana tabacum) resulted in plants with chlorotic leaves that accumulate soluble sugars and starch (Burkle et al., 1998), phenotypes resembling a tdy1 region. Future studies will investigate potential associations between TDY1 and maize SUTs in controlling carbohydrate accumulation in leaves. Understanding how Tdy1 regulates carbon partitioning may have applicability toward engineering biomass feedstocks for biofuels production.
Growth Conditions and Genetic Stocks
Maize (Zea mays) plants were grown at the Pennsylvania State University Rock Springs agronomy farm during the summers of 2005 and 2006. For studies of the boundaries of tdy1 regions, the tdy1-Reference (hereafter tdy1) mutation was introgressed nine times into the B73 inbred background. For all other experiments, tdy1 plants in a W23-derived stock capable of anthocyanin expression in leaves were used. In this line, anthocyanin specifically marked tdy1 regions (Braun et al., 2006 The albino w14 mutation was obtained from the Maize Genetics Cooperation Stock Center and maps to the long arm of chromosome 6. We fine mapped w14 3.6 cM proximal from bnlg1702 (two recombinants/56 chromosomes) and 10.7 cM distal to bnlg2249 (six recombinants/56 chromosomes), placing w14 approximately 45 cM proximal to tdy1. The crossing scheme used to generate stocks for the Tdy1 mosaic analysis is diagrammed in Supplemental Figure S1.
Transverse free-hand sections were cut from leaf material using a razor, mounted in water, and observed on a Nikon Eclipse 80i fluorescent microscope. Observations were made under bright field, then subsequently under UV using a 365-nm excitation filter and a 420-nm long pass emission filter, or fluorescein isothiocyanate using a 470-nm excitation filter and a 515-nm long pass emission filter, with epifluorescent illumination provided by a 100-W mercury lamp. To characterize starch accumulation in photosynthetic cells, sections were stained on the slide for approximately 10 s using a 1.25% (w/v) IKI solution (Ruzin, 1999
Sharp boundaries of tdy1 regions were examined to determine the frequency at which boundaries coincided with a particular class of veins. A region boundary was frequently discontinuous along its length, such that different parts might define more than one class. Frequency was defined as the percentage a particular class occurred among all boundaries scored. A total of 104 distinct boundaries from 25 tdy1 regions on 10 leaves was evaluated.
Leaves were decolorized by boiling in 95% ethanol, rinsed in water, stained in 1.25% IKI for approximately 5 min, rinsed, and photographed.
Maize seeds were imbibed in darkness for 45 h at room temperature on moist paper towels. For the Tdy1 mosaic analysis, 5,000 germinating seeds were irradiated. tdy1 stocks were constructed using the anthocyanin-accumulating genetic background to specifically mark tdy1 regions, as described previously (Braun et al., 2006
The following materials are available in the online version of this article.
We thank Tony Omeis, Scott Harkam, and Bob Oberheim for excellent plant care. We also thank Marty Sachs and Phil Stinard at the Maize Genetics Cooperative Stock Center for providing stocks. We are grateful to Candace Davison for irradiating seeds and Lauren Kawaguchi for assistance mapping the w14 locus. We appreciate the substantial improvements to the manuscript from two anonymous reviewers. We thank Surinder Chopra and Simon Gilroy for critically reading the article and members of the Braun and McSteen labs for discussions of the data and comments on the manuscript. Received March 4, 2007; accepted April 2, 2007; published April 13, 2007.
1 This work was supported by the National Research Initiative of the U.S. Department of Agriculture Cooperative State Research, Education and Extension Service (grant no. 2004–35304–14948 to D.M.B.).  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.plantphysiol.org) is: David M. Braun (dbraun{at}psu.edu).
[C] Some figures in this article are displayed in color online but in black and white in print.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.098814 * Corresponding author; e-mail dbraun{at}psu.edu; fax 814–865–9131.
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ABSTRACT
RESULTS
irradiation of germinating seeds induced chromosome breakage and uncovered albino, aneuploid w14 tdy1/– 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 (
