tie-dyed1 Regulates carbohydrate accumulation in maize leaves

Abstract

Acquisition of cell identity requires communication among neighboring cells. To dissect the genetic pathways regulating cell signaling in later leaf development, a screen was performed to identify mutants with chloroplast pigmentation sectors that violate cell lineage boundaries in maize (Zea mays) leaves. We have characterized a recessive mutant, tie-dyed1 (tdy1), which develops stable, nonclonal variegated yellow and green leaf sectors. Sector formation requires high light, occurs during a limited developmental time, and is restricted to leaf blade tissue. Yellow tdy1 sectors accumulate excessive soluble sugars and starch, whereas green sectors appear unaffected. Significantly, starch accumulation precedes chlorosis in cells that will become a yellow sector. Retention of carbohydrates in tdy1 leaves is associated with a delay in reproductive maturity, decreased stature, and reduced yield. To explain the tdy1 sectoring pattern, we propose a threshold model that incorporates the light requirement and the hyperaccumulation of photoassimilates. A possible function consistent with this model is that TDY1 acts as a sugar sensor to regulate an inducible sugar export pathway as leaves develop under high light conditions.

Figure 1.

Clonal and nonclonal sectors in maize leaves. A, Arrow indicates clonal sector of white tissue illustrating longitudinal arrangement of cell files in a maize leaf. B, Variegated tdy1 mutant leaf with yellow and green nonclonal sectors. C, tdy1 leaf in anthocyanin-accumulating genetic background showing that yellow sectors accumulate red anthocyanins. White circle shows a regional island sector located adjacent to tissue of the opposite sector type. D, tdy1 red sectors occur only in leaf blade tissues; leaf sheaths and husk leaf sheaths do not develop sectors. White arrow indicates a tdy1 sector in leaf blade tissue at the tip of an ear husk leaf.

Figure 2.

High light is required for tdy1 sector formation. A, Plants grown in the low light treatment (75 μmol m⁻² s⁻¹ light). B, Plants grown in the high light treatment (1,400 μmol m⁻² s⁻¹ light). C, Subsequent leaves produced after plants were transferred from low to high light. WT, Wild type.

Figure 3.

tdy1 sectors are evident 5 d after leaf emergence from whorl. A, Day 1, blade portion of the leaf has fully emerged from the whorl. B, Day 5, faint yellow-green sector is visible. C, Day 10, the sector has a lighter olive-green coloration compared to neighboring green tissue. D, Day 18, the sector is marked by anthocyanin accumulation. Asterisks in B, C, and D mark the tip of the sector.

Figure 4.

tdy1 yellow sectors have reduced membrane staining and decreased chlorophyll autofluorescence. A to C, Fluorescent micrographs of cross sections of mature leaves stained with DiOC₇, a dye that stains membranes. White arrowheads indicate dark precipitate. A, Wild-type leaf. Bundle sheath (BS) cells surround the veins and mesophyll (M) cells surround the BS cells. Black arrow indicates intensely fluorescent mesophyll cell chloroplast. B, tdy1 green sector likewise shows bright membrane staining in mesophyll chloroplasts. C, tdy1 yellow sector displays greatly reduced membrane staining and abundant dark precipitates in both bundle sheath and mesophyll cells. D to F, Bright-field illumination of free-hand cross sections. G to I, UV illumination of free-hand cross sections. D and G, Wild type. E and H, tdy1 green sector. F and I, tdy1 yellow sector. Scale bars = 10 μm (A–C); 50 μm (D–I).

Figure 5.

Photosynthetic cells in tdy1 yellow sectors hyperaccumulate starch. TEM micrographs show ultrastructure of mesophyll (M) and bundle sheath (BS) cells. Starch grains are visible as white crystals inside chloroplasts indicated with white arrows. Black arrows show grana stacks in mesophyll chloroplasts. A, Wild-type leaf showing starch accumulation in BS cell chloroplasts, but no starch accumulation in M cell chloroplasts. B, tdy1 green-leaf tissue showing starch accumulation in BS cell chloroplasts, but no starch in M cell chloroplasts. C, tdy1 yellow sectors hyperaccumulate starch grains in both M and BS cell chloroplasts. Scale bars = 5 μm.

Figure 6.

tdy1 yellow sectors accumulate starch throughout the sectors. A and B, Wild-type leaf. C and D, tdy1-sectored leaf. A and C, Photographs before staining. B and D, Photographs of cleared, IKI-stained leaves. [See online article for color version of this figure.]

Figure 7.

tdy1 yellow sectors accumulate excess soluble sugars and starch. Bars represent the mean of six samples and error bars represent the se. Wild type is represented in dark gray, tdy1 green sectors in light gray, and tdy1 yellow sectors in white. A, Suc. B, Glc. C, Fru. D, Starch quantification in leaves. An asterisk indicates that the value is significantly different from wild type at P ≤ 0.0001 using Student's t test. Units for each image are mg/g fresh weight.

Figure 8.

tdy1 yellow sectors accumulate starch prior to chlorosis. A and B, Wild-type leaf. C and D, tdy1 leaf. Leaves were divided into thirds corresponding to the tip (oldest), middle, and base (youngest) regions. A and C, Photographs before staining. B and D, Photographs of cleared, IKI-stained leaves. Arrows point to faintly visible yellow sector in mutant leaf tip that accumulates excess starch. Arrowhead indicates region of tissue preferentially accumulating starch prior to chlorosis.

Figure 9.

Reduced growth and yield in tdy1 mutants. Bars represent the mean and error bars represent the se. Wild type is represented in dark gray and tdy1 mutants in white. An asterisk indicates that the value is significantly different from wild type at P < 0.001 using Student's t test. A, tdy1 mutants have reduced plant height (n = 13). Units are centimeters above the soil surface. B, tdy1 mutants are delayed in flowering. Anthesis is pollen shed (n = 14) and silking is silk emergence (n = 15). Units are days after planting (dap). C, tdy1 mutants have reduced tassel size. D, tdy1 mutants have reduced ear size. WT, Wild type.