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. 2008 Mar;146(3):1085-97.
doi: 10.1104/pp.107.111476. Epub 2008 Jan 24.

Tie-dyed2 functions with tie-dyed1 to promote carbohydrate export from maize leaves

R Frank Baker  1 , David M Braun
Affiliations

Tie-dyed2 functions with tie-dyed1 to promote carbohydrate export from maize leaves

R Frank Baker et al. Plant Physiol. 2008 Mar.

Abstract

Regulation of carbon partitioning is essential for plant growth and development. To gain insight into genes controlling carbon allocation in leaves, we identified mutants that hyperaccumulate carbohydrates. tie-dyed2 (tdy2) is a recessive mutant of maize (Zea mays) with variegated, nonclonal, chlorotic leaf sectors containing excess starch and soluble sugars. Consistent with a defect in carbon export, we found that a by-product of functional chloroplasts, likely a sugar, induces tdy2 phenotypic expression. Based on the phenotypic similarities between tdy2 and two other maize mutants with leaf carbon accumulation defects, tdy1 and sucrose export defective1 (sxd1), we investigated whether Tdy2 functioned in the same pathway as Tdy1 or Sxd1. Cytological and genetic studies demonstrate that Tdy2 and Sxd1 function independently. However, in tdy1/+; tdy2/+ F(1) plants, we observed a moderate chlorotic sectored phenotype, suggesting that the two genes are dosage sensitive and have a related function. This type of genetic interaction is referred to as second site noncomplementation and has often, though not exclusively, been found in cases where the two encoded proteins physically interact. Moreover, tdy1; tdy2 double mutants display a synergistic interaction supporting this hypothesis. Additionally, we determined that cell walls of chlorotic leaf tissues in tdy mutants contain increased cellulose; thus, tdy mutants potentially represent enhanced feedstocks for biofuels production. From our phenotypic and genetic characterizations, we propose a model whereby TDY1 and TDY2 function together in a single genetic pathway, possibly in homo- and heteromeric complexes, to promote carbon export from leaves.

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Figures

Figure 1.
Figure 1.
tdy2 leaves exhibit chlorotic and green regions. A, Wild-type leaves have uniform green coloration. B, tdy1 leaves display variegated chlorotic and green regions. C, tdy2 leaves display a similar chlorotic and green variegated pattern. D, Hypoploid tdy2/− leaves show a mild chlorotic phenotype. E, Mature field-grown tdy2 plant displaying chlorotic leaves.
Figure 2.
Figure 2.
tdy2 chlorotic sectors hyperaccumulate carbohydrates. A and B, Photographs of wild-type and tdy2 leaf tissues, respectively. C and D, Photographs of cleared, starch-stained leaves in A and B showing that tdy2 chlorotic tissues hyperaccumulate starch relative to green tissues. E to H, Carbohydrate quantification in wild type (dark gray), tdy2 green tissues (light gray), and tdy2 chlorotic tissues (white). All units are milligram/gram fresh weight, and values represent averages of six samples ± se. Asterisks indicate values significantly different than wild type at P ≤ 0.01 determined using the Student's t test. E, Suc. F, Glc. G, Fru. H, Starch. [See online article for color version of this figure.]
Figure 3.
Figure 3.
Starch accumulation precedes chlorosis in tdy2 leaves. A and C, Photographs of emerging wild-type and tdy2 leaves, respectively. B and D, Leaves in A and C were cleared, starch stained, and photographed. tdy2 leaves contain regions that preferentially accumulated starch prior to visible chlorosis (arrows), while wild type did not show any differential accumulation of starch. [See online article for color version of this figure.]
Figure 4.
Figure 4.
Lateral veins frequently are found at sharp boundaries between tdy2 chlorotic and green tissues. A, Photograph of tdy2 leaf segment with sharp boundary between the green and the chlorotic region (arrow). B, Leaf cross section spanning the sharp boundary viewed in reflected light shows that a lateral vein (arrowhead) separates chlorotic and green tissues. C, Same section viewed in UV light shows reduced chlorophyll autofluorescence (red color) in the chlorotic tissue compared with the green tissue. Note that bundle sheath cells on either side of the lateral vein contain differing levels of chlorophyll. D, Same section starch stained and viewed in bright-field shows that mesophyll cells in chlorotic tissue contain starch (arrows). Additionally, bundle sheath cells on the chlorotic side of the lateral vein contain more starch than bundle sheath cells on the green side of the vein. Scale bars = 100 μm.
Figure 5.
Figure 5.
A mobile chloroplast by-product induces tdy2 phenotypic expression. A, ij1 leaf with longitudinal white stripes. B, tdy2 in anthocyanin expressing genetic background with red pigments accumulated solely in chlorotic regions. C, tdy2; ij1 double mutant leaf shows anthocyanin accumulation in albino cells adjacent to chlorotic, anthocyanin-accumulating tissues (arrow). D, Cleared, starch-stained leaf from C shows starch accumulates in albino, anthocyanin-accumulating cells (arrow). E to P, Leaf cross sections of each single mutant and tdy2; ij1 double mutant viewed in bright-field (E, H, K, and N) and under UV light (F, I, L, and O). G, J, M, and P show starch-stained sections in bright-field. Arrows in J and P show starch accumulation in mesophyll cells. E to G, tdy2 green tissue. H and J, tdy2 chlorotic tissue. K to M, ij1 albino tissue. N to P, tdy2; ij1 albino tissue accumulating anthocyanin. Dark color in N corresponds to air bubbles. Scale bars = 50 μm.
Figure 6.
Figure 6.
tdy2 leaf minor veins lack ectopic callose deposits. A, Cross section of minor vein viewed under UV light. Cells below black dotted line were removed to expose vascular interface (yellow dotted line). Arrow indicates orientation of view in B to E. BS, Bundle sheath; VP, vascular parenchyma; P, phloem; X, xylem. B to E, Paradermal sections of leaf minor veins stained with aniline blue and viewed in UV light. B, Wild type. Arrowhead indicates sieve plate. C, sxd1 mutants display ectopic callose deposits (arrow). D and E, tdy2 green and chlorotic tissues, respectively, lack ectopic callose deposits. Scale bars = 50 μm. [See online article for color version of this figure.]
Figure 7.
Figure 7.
tdy2 mutants have normal-appearing plasmodesmata at the BS-VP cell interface. TEM images of wild-type (A and C) and tdy2 chlorotic (B and D) tissues. BS, Bundle sheath; M, mesophyll; VP, vascular parenchyma. A, Wild-type bundle sheath chloroplasts contain abundant starch crystals, while mesophyll plastids contain little starch. B, tdy2 bundle sheath and mesophyll chloroplasts hyperaccumulate starch. Arrows in A and B indicate the BS-VP cell interface. C, Wild-type BS-VP cellular interface with plasmodesmata connecting the adjacent cytoplasms. D, tdy2 chlorotic tissue has normal appearing plasmodesmata between BS-VP cells. Arrows in C and D indicate the location of the plasma membrane in the BS cells. Scale bars in A and B = 5 μm; in C and D = 250 nm.
Figure 8.
Figure 8.
Genetic interaction between tdy1 and tdy2. A, C, and E, Leaves before staining. B, D, and F, Same leaves cleared and starch stained. A and B, Wild type. C and D, tdy1/+; tdy2/+ F1 leaf with chlorotic sectors. E and F, tdy1; tdy2 double mutant leaf displays complete chlorosis and starch hyperaccumulation throughout. G, Left to right, wild type, tdy2 single mutant, tdy1 single mutant, tdy1; tdy2 double mutant. [See online article for color version of this figure.]
Figure 9.
Figure 9.
tdy mutants deposit increased cellulose in their cell walls. A to D, Leaf cross sections stained with calcofluor white and viewed under UV light. A, Wild type. B, tdy2 chlorotic tissue. C, tdy1 chlorotic tissue. D, tdy1; tdy2 double mutant chlorotic tissue. Scale bars = 100 μm. [See online article for color version of this figure.]
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