Tie-dyed1 and sucrose export defective1 act independently to promote carbohydrate export from maize leaves

Abstract

tie-dyed1 (tdy1) and sucrose export defective1 (sxd1) are recessive maize (Zea mays) mutants with nonclonal chlorotic leaf sectors that hyperaccumulate starch and soluble sugars. In addition, both mutants display similar growth-related defects such as reduced plant height and inflorescence development due to the retention of carbohydrates in leaves. As tdy1 and sxd1 are the only variegated leaf mutants known to accumulate carbohydrates in any plant, we investigated whether Tdy1 and Sxd1 function in the same pathway. Using aniline blue staining for callose and transmission electron microscopy to inspect plasmodesmatal ultrastructure, we determined that tdy1 does not have any physical blockage or alteration along the symplastic transport pathway as found in sxd1 mutants. To test whether the two genes function in the same genetic pathway, we constructed F(2) families segregating both mutations. Double mutant plants showed an additive interaction for growth related phenotypes and soluble sugar accumulation, and expressed the leaf variegation pattern of both single mutants indicating that Tdy1 and Sxd1 act in separate genetic pathways. Although sxd1 mutants lack tocopherols, we determined that tdy1 mutants have wild-type tocopherol levels, indicating that Tdy1 does not function in the same biochemical pathway as Sxd1. From these and other data we conclude that Tdy1 and Sxd1 function independently to promote carbon export from leaves. Our genetic and cytological studies implicate Tdy1 functioning in veins, and a model discussing possible functions of TDY1 is presented.

Fig. 1

tdy1-R and sxd1-1 leaves display chlorotic sectors that hyperaccumulate carbohydrates. a Wild-type leaf showing uniform dark green color. b sxd1-1 mutant leaf displaying a chlorotic gradient, strongest at the tip to mildest toward the leaf base. Anthocyanins accumulate in the chlorotic tissues at the leaf tip and margins. c tdy1-R mutant leaf containing variegated chlorotic and green sectors throughout the leaf blade. d–f Cleared, IKI stained leaf tissues showing that mutant chlorotic regions hyperaccumulate starch. d Wild type. e sxd1-1. f tdy1-R. Scale bars represent 4 cm

Fig. 2

Aniline blue staining of ectopic callose deposits in leaf minor veins. a Cross section of a wild-type minor vein shown by UV autofluorescence indicating the cell types and orientation of view (arrow) for b–e. Abaxial cells were removed below the black dotted line to view the bundle sheath–vascular parenchyma cell interface. X xylem, P phloem, VP vascular parenchyma, BS bundle sheath, M mesophyll. b–eAniline blue fluorescence images of paradermal sections along minor veins. b Wild-type cells lack punctate callose deposits indicating no blockages along the symplastic pathway. The bright fluorescence observed in the phloem corresponds to sieve plates (arrowhead). c sxd1-1 minor veins contain many callose deposits at the bundle sheath–vascular parenchyma cell interface (arrow). d tdy1-R chlorotic tissue lacks callose deposition over plasmodesmata in any cells. e tdy1-R; sxd1-1 double mutants contain callose deposits (arrow) at the bundle sheath–vascular parenchyma cell interface comparable to the level seen in sxd1-1 single mutants. Scale bar in a represents 50 μm, and in b–e 10 μm

Fig. 3

TEM images of cellular interfaces along the symplastic pathway of minor veins. a-d Bundle sheath–vascular parenchyma cells. Arrows indicate the location of the plasma membrane in the bundle sheath cell. a Wild-type plasmodesmata span the cell wall, lack occlusions, and connect with the plasma membrane. b sxd1-1 chlorotic leaf tips contain occlusions over the plasmodesmata on the bundle sheath cell side of the cell wall. c tdy1-R chlorotic sectors contain normal appearing plasmodesmata that lack occlusions. Note the plasmodesmata span the cell wall and connect with the plasma membrane. d tdy1-R; sxd1-1double mutant chlorotic tissues contain deposits over the plasmodesmata in the bundle sheath cell similar to sxd1-1 single mutants. Additional cellular interfaces along the symplastic pathway of wild type (e, g, i) and tdy1-R chlorotic tissues (f, h, j) are shown. In e–j the plasmodesmata spanned the cell wall connecting the cytoplasms of adjacent cells and lacked occlusions. e, f BS–BS cells, g, h BS–M cells, i, j M–M cells. BS bundle sheath, M mesophyll. Scale bars represent 200 nm

Fig. 4

Phenotypic comparisons among plants from a F₂ family segregating tdy1-R and sxd1-1 mutants. In a and c the order from left to right corresponds to wild type, tdy1-R, sxd1-1, and tdy1-R; sxd1-1 double mutant. a Double mutants show the greatest reduction in plant height compared with either single mutants or wild-type siblings. b Double mutant leaves are severely chlorotic with lighter green tissues restricted to the leaf base. Distinct borders between the chlorotic and green tissues characteristic of tdy1-R are visible at the leaf base. The strong tip to base chlorosis gradient of sxd1-1 expression is evident. c Tassel height is most strongly reduced in tdy1-R; sxd1-1 double mutants compared with sxd1-1 and tdy1-R single mutants or wild-type siblings. d Approximately half of the tdy1-R; sxd1-1 double mutant plants produced barren tassels lacking spikelets. Scale bars represent 4 cm

Fig. 5

Carbohydrate quantification in leaves of wild-type, tdy1-R, sxd1-1 and double mutant plants. Units for all panels are mg carbohydrate/g fresh weight. Values are the means of 18 samples ± SE. Abbreviations are wt, wild-type, dm, tdy1-R; sxd1-1 double mutant. Different letters indicate statistically significant differences between samples determined using Student’s t test

Fig. 6

Model for TDY1 functions in phloem loading of sucrose. Diagrams represent the pathway of sucrose movement from photosynthetic cells into the phloem. M mesophyll, BS bundle sheath, VP vascular parenchyma, CC companion cell, SE sieve element. Green and yellow ovals in M and BS cells represent chloroplasts. White circles within chloroplasts represent starch grains. Magenta box on VP plasma membrane represents sucrose efflux transporter, and blue boxes on CC and SE plasma membranes depict sucrose transporters. Arrows indicate the direction of sucrose movement. a Wild-type tissue with normal, unimpeded sucrose movement into the phloem. b sxd1-1 mutant chlorotic tissue has callose deposits (red box) over the plasmodesmata at the BS–VP cell interface blocking sucrose movement and resulting in carbohydrate accumulation in M and BS cells. c tdy1-R chlorotic tissue has normal appearing plasmodesmata and lacks callose deposits. We hypothesize that either the sucrose efflux transport step is inhibited (red X) or the CC–SE sucrose transporters (orange X) fail to load sucrose into the phloem. This would result in the carbon hyperaccumulation observed in the photosynthetic cells