Tie-dyed1 and Sucrose export defective1 act independently to promote carbohydrate export from maize leaves tie-dyed1 -LRB- tdy1 -RRB- and sucrose export defective1 -LRB- sxd1 -RRB- are recessive maize -LRB- Zea mays -RRB- 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 F2 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 . Introduction Sugars synthesized in photosynthetic leaf cells must be transported into the veins for distribution to nonphotosynthetic tissues . Maize -LRB- Zea mays -RRB- is a C4 plant which displays Kranz anatomy in its leaves -LRB- Esau 1977 -RRB- . Veins are surrounded by bundle sheath cells which in turn are surrounded by mesophyll cells . Mesophyll and bundle sheath cells cooperatively perform the reactions of photosynthesis and carbon assimilation . Sucrose is synthesized in the cytoplasm of mesophyll cells -LRB- Lunn and Furbank 1999 -RRB- , diffuses through plasmodesmata into bundle sheath cells , and then into vascular parenchyma cells -LRB- Russin et al. 1996 -RRB- . Maize is an apoplastic phloem loading species -LRB- Evert et al. 1978 -RRB- , whereby sucrose is exported from the vascular parenchyma cells to the apoplast by an unknown mechanism -LRB- see Lalonde et al. 2004 for discussion -RRB- . Sucrose is then imported into phloem companion cells and/or sieve elements by sucrose transporters located in the plasma membrane -LRB- Aoki et al. 1999 ; Lalonde et al. 2004 ; Sauer 2007 ; Scofield et al. 2007 -RRB- . The energy to transport sucrose into the phloem is derived from H + - ATPases that generate the proton motive force across the plasma membrane -LRB- Bush 1993 ; Gaxiola et al. 2007 -RRB- . In addition , the abundance of the sucrose transporter that controls phloem loading is transcriptionally regulated by a sucrose-sensing signal transduction pathway -LRB- Chiou and Bush 1998 ; Vaughn et al. 2002 ; Ransom-Hodgkins et al. 2003 -RRB- . If carbon transport into the vein is blocked , carbohydrates accumulate in the photosynthetic cells , leading to down-regulation of photosynthetic gene expression and reduced chlorophyll levels -LRB- chlorosis -RRB- -LRB- Sheen 1990 ; Goldschmidt and Huber 1992 ; Riesmeier et al. 1994 ; Krapp and Stitt 1995 ; Burkle et al. 1998 ; Gottwald et al. 2000 ; Jeannette et al. 2000 -RRB- . Even though the transport pathway for assimilated carbon has been studied in maize leaves for almost 40 years -LRB- Hofstra and Nelson 1969 -RRB- , the mechanisms that regulate sucrose export from leaves remain poorly understood . To date , two recessive maize mutants have been described with defects in carbon export from leaves . tie-dyed1 -LRB- tdy1 -RRB- and sucrose export defective1 -LRB- sxd1 -RRB- mutants develop nonclonal chlorotic leaf sectors that hyperaccumulate starch and soluble sugars -LRB- Russin et al. 1996 ; Provencher et al. 2001 ; Braun et al. 2006 -RRB- . Other mutants with variegated leaves have been characterized , but none have carbohydrate accumulation within the chlorotic regions -LRB- Rhoades and Carvalho 1944 ; Fisher and Eschrich 1985 ; Madore 1990 ; Yu et al. 2007 -RRB- . tdy1 and sxd1 both display growth-related defects such as reduced plant height and inflorescence development due to the retention of carbohydrates in leaf tissues . However , their phenotypes also show significant differences . sxd1 mutant leaves progressively exhibit chlorosis which initiates at the leaf tip and basipetally spreads toward the base . Interestingly , in sxd1 mutants , only the leaf minor veins are affected in phloem loading -LRB- Russin et al. 1996 -RRB- . The tdy1 mutant phenotype is distinct from sxd1 mutants in that large chlorotic regions develop throughout leaf blade tissue . Further , tdy1 chlorotic regions form only during a limited time as the leaf emerges from the whorl and , once formed , do not expand -LRB- Braun et al. 2006 -RRB- . Lastly , in tdy1 mutants , chlorotic tissues are often bounded by lateral veins , implicating this vein class in limiting the expansion of a tdy1 chlorotic sector -LRB- Baker and Braun 2007 -RRB- . Sxd1 encodes tocopherol cyclase , an enzyme functioning in tocopherol -LRB- vitamin E -RRB- biosynthesis , and sxd1 mutants lack tocopherols -LRB- Sattler et al. 2003 -RRB- . Absence of tocopherols results in callose deposition over the plasmodesmata at the bundle sheath-vascular parenchyma cell interface of leaf minor veins -LRB- Russin et al. 1996 ; Botha et al. 2000 ; Provencher et al. 2001 -RRB- . This occludes these passageways and prevents sucrose from moving into the vein , resulting in the build-up of carbohydrates in photosynthetic cells . The function of tocopherols in preventing callose deposition in the phloem is evolutionarily conserved . RNAi-mediated suppression of tocopherol cyclase activity in potato results in callose deposition in vascular-associated cells and carbohydrate accumulation in the photosynthetic cells -LRB- Hofius et al. 2004 -RRB- . In addition , in Arabidopsis tocopherol cyclase mutants and other vitamin E deficient mutants , callose is deposited in phloem parenchyma transfer cells and the photosynthetic cells accumulate carbohydrates if the plants are subjected to nonfreezing low temperatures -LRB- Maeda et al. 2006 -RRB- . It is not known how lack of tocopherols causes callose deposition . Because both mutants show defects in carbon export from leaves , we examined whether Tdy1 and Sxd1 function in the same pathway to limit leaf carbohydrate accumulation . Using aniline blue fluorescence microscopy and transmission electron microscopy -LRB- TEM -RRB- we determined that carbohydrate accumulation in tdy1 mutant leaves does not result from a similar impediment to phloem loading as in sxd1 mutants . To genetically test whether the two genes function in the same pathway , we analyzed F2 families segregating both mutations . We observed that the double mutant plants showed an additive interaction for growth-related phenotypes and soluble sugar accumulation indicating that the two genes function in separate genetic pathways . To determine if Tdy1 functions in the same biochemical pathway as Sxd1 , we measured tocopherol levels in mutant plants . We found that tdy1 mutants contain wild-type levels of tocopherols , indicating that Tdy1 does not function in the same biochemical process as Sxd1 . Thus , from the combination of cellular , genetic and biochemical investigations we conclude that Tdy1 and Sxd1 function independently to promote carbon export from leaves . Additionally , genetic and cytological studies suggest that Tdy1 functions within the veins , and a model of possible TDY1 functions is discussed . Materials and methods Plant materials Plants were grown in the summer in the Rock Springs Agronomy Farm , Pennsylvania State University , and in the winter in Juana Diaz , Puerto Rico . The tdy1-Reference -LRB- tdy1-R -RRB- -LRB- Braun et al. 2006 -RRB- and sxd1-1 -LRB- Russin et al. 1996 -RRB- mutations were backcrossed to the B73 inbred line five times prior to crossing together . F2 families were generated by crossing tdy1-R and sxd1-1 mutant plants and self-pollinating the F1 individuals . Morphological analyses Plant height was measured from the soil surface to the tip of the central spike of the tassel . Tassel length was measured from the node of the flag leaf to the tip of the central spike . Anthesis was recorded as the day of first pollen shed . Plants were scored as having produced ears if either -LRB- 1 -RRB- ears were clearly visible , -LRB- 2 -RRB- silks were visible emerging from the leaf sheath , or -LRB- 3 -RRB- ears developed such that they were identifiable by peeling back the ear leaf sheath . For wild-type and tdy1-R single mutants n = 18 for all measurements . For sxd1-1 single mutants , 12 of the 16 plants produced a tassel that shed pollen , and 13 produced an ear . For tdy1-R ; sxd1-1 double mutants , 7 of the 15 plants shed pollen and 8 made an ear . Barren tassels that did not shed pollen were not included in the tassel length or anthesis measurements . Aniline blue staining Leaf strips 1 cm in width were cut from mutant and wild-type leaves . The abaxial epidermis and the mesophyll cells directly beneath it were gently scraped away with a single-edge razor to expose the inner leaf tissues . The leaf strip was placed on a slide , briefly stained with a 0.05 % aniline blue and immediately viewed using a 360 -- 370-nm excitation filter and a 420-nm long pass emission filter on a Nikon Eclipse 80i fluorescent microscope . Epifluorescent illumination was provided by a 100-W mercury lamp , and images were recorded using a DXM1200F Nikon digital camera . TEM analyses For tissue fixation , leaf samples were diced into 3 × 1 mm pieces , placed in 4 % glutaraldehyde , 1 % paraformaldehyde , 0.3 % Tween 20 , 50 mM sodium cacodylate , pH 7.4 , and vacuum infiltrated on ice for 4 -- 6 h. Samples were post-fixed in 1 -- 2 % osmium tetroxide at 4 °C overnight , subsequently dehydrated through a graded acetone series and embedded in Spurr 's epoxy resin -LRB- Spurr 1969 -RRB- . Thin sections -LRB- 90 -- 100 nm -RRB- were cut on a Leica Ultracut E ultramicrotome with a glass knife and lifted onto 200-mesh copper grids . The grids were stained in 2 % uranyl acetate , followed by Reynold 's lead citrate and observed with a JEOL JEM 1200 EXII at an accelerating voltage of 80 kV . Chlorophyll quantification Relative levels of total chlorophylls were quantified from leaves using a SPAD 502 Data Logger Chlorophyll Meter -LRB- Spectrum Technologies -RRB- . For each tissue , 30 samples were measured , and the experiment was performed three times . Representative data from one replicate are shown . Sugars and starch quantification Leaf tissues were collected at the end of the photoperiod , weighed and frozen at − 80 °C . Carbohydrate extraction was performed according to Dinges et al. -LRB- 2001 -RRB- . Sugars and starch were quantified using commercial assay kits according to the manufacturer 's instructions -LRB- R-Biopharm -RRB- . Six samples from each tissue type were measured in triplicate . For visualizing starch in leaves , samples were collected at the end the photoperiod , decolored by boiling in 95 % ethanol and stained with iodine-potassium iodide -LRB- IKI -RRB- -LRB- Ruzin 1999 -RRB- . Tocopherol quantification Leaf discs of 11 mm were harvested with a cork borer from leaf tissues , weighed and immediately frozen on dry ice . Total lipids were extracted from collected tissues and tocopherols measured according to -LRB- Sattler et al. 2003 -RRB- . Results tdy1-R and sxd1-1 single mutants both display variegated chlorotic leaves tdy1-R and sxd1-1 mutants show striking parallels in their leaf phenotypes . Both develop chlorotic sectors that hyperaccumulate starch and soluble sugars in leaf blades -LRB- Fig. 1 -RRB- . However , there are also distinctions . Whereas wild-type leaves have a uniform , dark green color , sxd1-1 mutant leaves develop a chlorotic phenotype that shows a continuum of expression from strongest at the tip to mildest toward the base -LRB- Fig. 1a , b -RRB- . In addition , at the tip and leaf margins , the chlorotic tissues progressively accumulate anthocyanin and excess starch -LRB- Fig. 1b , e -RRB- . In sxd1-1 leaves , no clear , delineated boundaries occur between green and chlorotic regions . Conversely , tdy1-R mutants display a variegated pattern of chlorotic and normal appearing green regions throughout leaf blade tissue -LRB- Fig. 1c -RRB- . Additionally , the boundaries between tdy1-R chlorotic and normal appearing green tissue are sharp and distinct , and they tend to occur at lateral veins -LRB- Baker and Braun 2007 -RRB- . As in sxd1-1 mutants , chlorotic regions in tdy1-R mutants hyperaccumulate starch relative to green tissues -LRB- Fig. 1f -RRB- . tdy1 and sxd1 are the only known leaf variegation mutants that hyperaccumulate carbohydrates in any plant ; therefore , we investigated whether Tdy1 and Sxd1 function in a common pathway influencing carbon partitioning in leaves . Fig. 1tdy1-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 Carbohydrate accumulation in tdy1-R mutants occurs by a different mechanism than in sxd1-1 plants To address whether the two genes function in the same pathway , we investigated if the mechanism for carbohydrate accumulation was similar in the two mutants . Carbohydrate accumulation in sxd1-1 mutants results from a blockage in the symplastic pathway of sucrose movement -LRB- Russin et al. 1996 ; Botha et al. 2000 ; Provencher et al. 2001 -RRB- . Specifically , at the bundle sheath -- vascular parenchyma cell interface of leaf minor veins , callose is deposited over the plasmodesmata in bundle sheath cells . This blockage prevents sucrose from being loaded into the phloem and results in carbohydrate accumulation in the photosynthetic cells . To determine if the tdy1-R leaf phenotype is similarly caused by callose deposits plugging the plasmodesmata , we performed aniline blue staining and fluorescence microscopy . Aniline blue binds callose and fluoresces a blue -- white color under UV light . In wild-type plants , only the sieve plates in the end walls of phloem sieve elements stain positively for callose -LRB- Fig. 2b -RRB- . As previously reported , sxd1-1 mutants display many punctate callose deposits at the vascular parenchyma-bundle sheath cell interface of minor veins -LRB- Fig. 2c -RRB- -LRB- Botha et al. 2000 -RRB- . In tdy1-R chlorotic regions , no ectopic callose deposits were detected at this interface -LRB- Fig. 2d -RRB- . Further , no callose deposits were observed in any cells of minor or lateral veins in tdy1-R chlorotic tissues , nor in any cells of tdy1-R green regions -LRB- data not shown -RRB- . These data suggest that tdy1-R mutants do not accumulate carbohydrates due to callose blocking symplastic sucrose movement . Fig. 2Aniline 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 -LRB- arrow -RRB- 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 -LRB- arrowhead -RRB- . c sxd1-1 minor veins contain many callose deposits at the bundle sheath -- vascular parenchyma cell interface -LRB- arrow -RRB- . d tdy1-R chlorotic tissue lacks callose deposition over plasmodesmata in any cells . e tdy1-R ; sxd1-1 double mutants contain callose deposits -LRB- arrow -RRB- 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 Although we did not observe ectopic callose deposition in any cells in tdy1-R mutant leaves using aniline blue staining , it is possible that symplastic sucrose transport is precluded due to a different defect in plasmodesmatal structure . To investigate this possibility , we performed TEM to inspect the plasmodesmata ultrastructure along the symplastic pathway . In minor veins from wild-type leaves , the plasmodesmata between bundle sheath and vascular parenchyma cells were unobstructed , spanned the cell wall and connected the cytoplasms between the two cells -LRB- Fig. 3a -RRB- . Consistent with previous reports , minor veins from leaf tips expressing the sxd1-1 mutant phenotype showed occlusions over the plasmodesmata on the bundle sheath cell side of the bundle sheath -- vascular parenchyma cell interface -LRB- Fig. 3b -RRB- -LRB- Russin et al. 1996 ; Botha et al. 2000 ; Provencher et al. 2001 -RRB- . In tdy1-R mutant leaves , we found no occlusions over the plasmodesmata or alterations in their structure between the bundle sheath and vascular parenchyma cells in minor veins -LRB- Fig. 3c -RRB- . In examining plasmodesmata along the symplastic pathway at all cellular interfaces in tdy1-R leaves , we did not observe any structural perturbations or occlusions -LRB- Fig. 3e -- j -RRB- . These data indicate that carbohydrate retention in tdy1-R mutant leaves does not appear to result from physical blockage or plasmodesmatal structural changes . Together , the aniline blue staining and TEM studies indicate that the cellular basis for the tdy1-R and sxd1-1 phenotypes is different . Fig. 3TEM 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 -LRB- e , g , i -RRB- and tdy1-R chlorotic tissues -LRB- f , h , j -RRB- 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 Double mutant plants show more severe growth defects than either single mutant Both tdy1-R and sxd1-1 single mutant plants have growth-related defects due to the retention of carbohydrates in leaves . To determine whether the two genes function in the same genetic pathway promoting carbon export from leaves , we created F2 families segregating both mutations . Both mutations are recessive and the F2 segregation ratio fits the expected 9:3:3:1 prediction , indicating that neither mutant shows epistasis -LRB- Table 1 -RRB- . sxd1-1 is a molecular null allele lacking transcripts -LRB- Provencher et al. 2001 -RRB- , and based on dosage analysis , tdy1-R is a genetic null allele -LRB- Braun et al. 2006 -RRB- . If Tdy1 and Sxd1 act in a linear genetic pathway , we would expect that the phenotype of the double mutant would be similar to one or the other mutant . If the two genes function in separate pathways , then we predict that the double mutant would be more severe than either single mutant . Table 1Genetic segregation in F2 familiesFamily #TotalWild typetdy1-Rsxd1-1tdy1-R ; sxd1-1χ2DB 55039348221672.0 P > 0.05 DB 55129552231552.1 P > 0.05 DB 55239355181550.6 P > 0.05 Total2811556346172 .9 P > 0.05 χ2 analyses of tdy1-R and sxd1-1 segregation in three F2 families conform to a 9:3:3:1 expectation . Observed numbers of individuals are shown To examine the interactions between tdy1-R and sxd1-1 , we quantified the growth parameters of plant height , tassel size , time to anthesis and ear production -LRB- Fig. 4 , Table 2 -RRB- . At the whole plant level , the double mutants express a more severe phenotype and show a stronger reduction in growth characteristics than either single mutant -LRB- Fig. 4a -RRB- . Compared to wild-type , tdy1-R single mutants have an 11 % decrease , sxd1-1 mutants have a 24 % reduction , and the tdy1-R ; sxd1-1 double mutant plants show the greatest reduction in plant height of 35.5 % . Similar to plant height , tassel size is reduced 13 % in tdy1-R plants , 29 % in sxd1-1 mutants , and 50 % in tdy1-R ; sxd1-1 double mutants -LRB- Fig. 4c , Table 2 -RRB- . In addition , approximately half of the tdy1-R ; sxd1-1 double mutant plants produced very reduced tassel branches lacking spikelets -LRB- Fig. 4d -RRB- . This phenotype was observed in 25 % of the sxd1-1 single mutants . Correlated with the tassel height defects , both mutations retarded time to flowering . Both tdy1-R and sxd1-1 single mutants first shed pollen eight days later than wild-type siblings -LRB- Table 2 -RRB- . Double mutant plants were delayed even further by approximately another 5 days . Similarly , wild-type and tdy1-R mutant plants invariably made ears , and sxd1-1 mutants produced ears 81 % of the time . In contrast , only 53 % of the double mutant plants produced an ear -LRB- Table 2 -RRB- . All of the growth defects observed in the single and double mutants are consistent with a restriction in assimilates transported to the growing portions of the plants . The double mutant plants showed the most severe phenotypes suggesting they have a greater reduction in carbohydrates exported out of leaves than in either single mutant . In support of this , the leaves of tdy1-R ; sxd1-1 double mutant plants are more strongly chlorotic than either single mutant , with a corresponding reduction in the amount of green tissue -LRB- compare Figs. 4b with 1b , c -RRB- . In double mutant leaves , the tip to base gradient of chlorosis and anthocyanin pigmentation characteristic of the sxd1-1 phenotype is superimposed on the tdy1-R chlorotic and green sectored pattern . Double mutant leaves still contain some green tissues located at the leaf base as in sxd1-1 single mutants ; however , the boundaries between the chlorotic and green regions are sharp and distinct as in tdy1-R mutants -LRB- Fig. 4b -RRB- . Thus , the double mutant leaves express the phenotypes of both single mutants , and double mutant plants display stronger growth retardation than either single mutant . Fig. 4Phenotypic 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 cmTable 2Morphometric analyses in F2 familiesPhenotypePlant heightwt % Tassel lengthwt % AnthesisEars -LRB- % -RRB- Wild type220 .4 a ± 0.3910055.2 a ± 0.1410071.9 a ± 0.06 100tdy1-R196.8 b ± 0.9289.247.9 b ± 0.28 86.880.3 b ± 0.13 100sxd1-1168.9 c ± 1.2476.639.3 c ± 0.1871.280.3 b ± 0.0981tdy1-R ; sxd1-1142.2 d ± 0.7964.526.8 d ± 0.34 48.684.9 c ± 0.1353 Values are the means ± SE . Plant height and tassel length are measured in cm . Anthesis is measured as the number of days after planting until first pollen shed . Percent ears indicates the frequency each phenotypic class produced an ear . Different letters in superscript within a column denote statistical significance determined using Student 's t test Since double mutants have more extensive chlorosis in their leaves , we ascertained whether the callose deposits at the vascular parenchyma-bundle sheath cell interface observed in sxd1-1 single mutants were more severe in the double mutants . Using aniline blue fluorescence , the double mutants showed no enhancement of the callose deposition phenotype and appeared similar to sxd1-1 single mutants -LRB- Fig. 2c , e -RRB- . Similarly using TEM , we found blockages over the plasmodesmata on the bundle sheath cell side of the bundle sheath -- vascular parenchyma cell interface of minor veins in the double mutants comparable to what we observed in sxd1-1 single mutant leaves -LRB- Fig. 3b , d -RRB- . In neither case was there any increase in the severity of the phenotype . Hence , tdy1-R and sxd1-1 do not display a synergistic interaction as the double mutant leaves express both single mutant phenotypes . Rather , these data suggest that the phenotypes observed in the double mutants are additive , and that the genes act in independent pathways . Leaf tips of double mutant plants have the greatest reduction in chlorophyll levels tdy1-R and sxd1-1 single mutants both show variegated chlorotic regions in their leaves -LRB- Fig. 1b , c -RRB- . We quantified total chlorophyll levels to determine whether the double mutants were more strongly affected than either single mutant -LRB- Table 3 -RRB- . We observed a decreasing series in chlorophyll content with the strongest reduction in the tips of double mutant leaves < the chlorotic regions of either tdy1-R or sxd1-1 single mutant leaves < the green base of sxd1-1 or double mutant leaves < green regions of tdy1-R mutants or wild-type leaves . The leaf bases of sxd1-1 mutants and the double mutants displayed a mild chlorosis phenotype with approximately 70 % as much chlorophyll as wild-type leaves indicating that these tissues are affected by the sxd1-1 mutation . As the tips of double mutant leaves showed a more severe chlorosis phenotype than either single mutant , it suggests that Tdy1 and Sxd1 function in separate pathways . Table 3Chlorophyll quantification in F2 plantsPhenotypeChlorophyllWild type55 .7 a ± 0.55tdy1-R green54 .8 a ± 0.57sxd1-1 green41 .0 b ± 0.75tdy1-R ; sxd1-1 green42 .2 b ± 0.67tdy1-R chlorotic20 .9 c ± 0.62sxd1-1 chlorotic21 .5 c ± 0.66tdy1-R ; sxd1-1 chlorotic11 .4 d ± 0.64 Chlorophyll levels were measured in different phenotypic -LRB- green or chlorotic -RRB- tissues from different genotypes . Values represent the means ± SE in relative units -LRB- n = 30 -RRB- . Statistical significance was determined using Student 's t test and indicated by different letters in superscript Sugar accumulation in double mutant leaves is greater than in either single mutant Both single mutants accumulate excess starch in the chlorotic regions of their leaves -LRB- Fig. 1e , f -RRB- . To investigate whether Tdy1 and Sxd1 act in the same or separate genetic pathways limiting carbohydrate accumulation in leaves , we determined the starch and soluble sugars content in different phenotypic regions of F2 plants -LRB- Fig. 5 ; Table S1 in electronic supplementary material -RRB- . For both the green regions and the chlorotic regions , we found that the double mutants contained similar amounts of starch as the single mutants . In contrast , we determined that the levels of soluble sugars differed with respect to genotype and phenotype . As previously reported , we found green regions of tdy1-R leaves had sucrose , glucose and fructose levels indistinguishable from wild type -LRB- Braun et al. 2006 -RRB- . However , green leaf bases of the double mutants and sxd1-1 single mutants contained approximately fourfold higher levels of glucose and fructose as wild type or tdy1-R green tissues . Among the chlorotic leaf tissues , the double mutant leaf tips had the highest concentrations of sugars , with approximately 33 % greater amounts of glucose , and a significant increase in sucrose content compared with either single mutant . Double mutant leaf tips also contained ∼ 30 % more fructose than sxd1-1 leaf tips and approximately two-fold greater levels than in tdy1-R chlorotic regions . Overall , sugar concentrations in green tissues of double mutant leaves are more similar to sxd1-1 leaf bases than to tdy1-R green regions , whereas in chlorotic tissues , the double mutants contain more soluble sugars than either single mutant . Because the double mutants show greater individual sugar accumulation than either single mutant , these data support the hypothesis that Tdy1 and Sxd1 function in independent pathways promoting carbon export from leaves . Fig. 5Carbohydrate 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 Tdy1 does not function in the same biochemical pathway as Sxd1 Sxd1 encodes tocopherol cyclase , the penultimate enzyme in the vitamin E biosynthesis pathway , and sxd1 mutants lack tocopherols -LRB- Sattler et al. 2003 -RRB- . To determine whether Tdy1 functions in the same biochemical pathway as Sxd1 , we quantified tocopherol levels in tdy1-R plants -LRB- Table 4 -RRB- . We found no statistically significant changes in tocopherol quantities in tdy1-R chlorotic or green regions in comparison to wild-type tissue . sxd1 mutants had undetectable levels of tocopherols as previously reported -LRB- Sattler et al. 2003 -RRB- . Hence , tdy1-R does not have a lesion in tocopherol synthesis or accumulation , and Tdy1 does not function in the same biochemical pathway as Sxd1 . Table 4Tocopherol quantificationPhenotypeTocopherolWild type118 .6 a ± 3.59tdy1-R green101 .0 a ± 3.17tdy1-R chlorotic112 .3 a ± 6.17sxd1-1ndValues are means of six samples ± SE measured in μg/g fresh weight . nd indicates none detected . Values indicated by letter `` a '' in superscript were not statistically different as determined using Student 's t test Discussion Tdy1 and Sxd1 independently promote carbon export in maize leaves tdy1 and sxd1 are the only known mutations in plants that confer a variegated leaf phenotype with sectors that hyperaccumulate carbohydrates . Using a combination of biochemical , cytological and genetic analyses we investigated whether Tdy1 and Sxd1 act in a common pathway . Whereas sxd1 mutants lack tocopherol , we determined that tdy1-R mutants accumulate wild-type levels of tocopherols . Therefore , Tdy1 does not function in the same biochemical process as Sxd1 . sxd1 mutants accumulate carbohydrates in leaf tissues due to callose being deposited over the interface of bundle sheath-vascular parenchyma cells in leaf minor veins which blocks sucrose symplastic movement . Using aniline blue fluorescence microscopy we did not identify any ectopic callose deposition in tdy1-R mutant leaf tissues . Moreover , using TEM we did not observe any structural alterations to the plasmodesmata , indicating that the symplastic loading pathway is not physically impeded . This suggests that tdy1-R mutants do not accumulate carbohydrates due to an occlusion , but rather due to a different cellular mechanism . This was confirmed by double mutant analyses which showed that the two mutations had an additive interaction . We found that the double mutants expressed both single mutant leaf phenotypes , that the reductions in plant height , tassel size and chlorophyll levels were additive , and that the accumulation of sugars in chlorotic leaf tissues of the double mutants was greater than in either single mutant . In addition , half of the double mutants produced ears whereas the tdy1-R mutant frequency was indistinguishable from wild type and 81 % of the sxd1-1 single mutants made ears . The increased frequencies in the failure to make ears and production of barren tassels observed in the double mutants are most likely explained by an additive retention of carbohydrates in leaf tissues and a concomitant failure to export sucrose to the developing inflorescences . Collectively , these data lead us to conclude that Tdy1 and Sxd1 function independently , and that tdy1 defines a distinct genetic pathway affecting carbon partitioning in maize leaves . Chlorotic leaf tissues do not retain sink identity We previously suggested that one possibility to explain the excess carbon accumulation in tdy1-R chlorotic leaf sectors was a block in the developmental transition from sink to source identity , such that the chlorotic sectors continued to import photoassimilates from neighboring green tissues -LRB- Braun et al. 2006 -RRB- . If the chlorotic tissues were to remain sinks , the large amounts of green source tissues could potentially provide the carbohydrates accumulating in the chlorotic regions . However , based on our genetic analyses , our data do not support this hypothesis . In the tdy1-R ; sxd1-1 double mutants , the amount of green leaf tissue is greatly reduced and is restricted to a narrow region at the very base of the leaves . Additionally , this green tissue displays mild chlorosis and an increase in carbohydrate accumulation indicating that it is perturbed in carbon export . As this tissue is proximal to and up to a meter distant from the chlorotic tip of adult leaves , it is highly unlikely to be providing the carbohydrates that accumulate in the cells near the tip . Furthermore , half of the double mutant plants produced a tassel and an ear . These are sink tissues dependent on the photosynthetic source leaves for their carbohydrates . Because the great majority of the area of double mutant leaves is severely chlorotic , yet still must be capable of exporting photoassimilates to produce the developing reproductive tissues , our data argues against the chlorotic tissues remaining sinks and does not support the hypothesis that Tdy1 functions to regulate the sink to source transition . Rather , the new data lead us to favor the hypothesis that the cells in the tdy1 chlorotic region are source tissues that are partly blocked in carbon export capacity . A model for TDY1 function In wild-type maize leaves , sucrose is synthesized in mesophyll cells , diffuses through plasmodesmata into bundle sheath cells and then into the vascular parenchyma cells -LRB- Fig. 6a -RRB- . Sucrose is exported from the vascular parenchyma cell to the apoplast , and then loaded into the phloem by sucrose transporters in the plasma membrane of the companion cell and/or sieve element -LRB- Lalonde et al. 2004 ; Sauer 2007 -RRB- . sxd1 mutant leaf tips are blocked in this pathway at the bundle sheath -- vascular parenchyma cell interface -LRB- Fig. 6b -RRB- -LRB- Russin et al. 1996 ; Botha et al. 2000 ; Provencher et al. 2001 -RRB- . Fig. 6Model 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 -LRB- red box -RRB- 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 -LRB- red X -RRB- or the CC -- SE sucrose transporters -LRB- orange X -RRB- fail to load sucrose into the phloem . This would result in the carbon hyperaccumulation observed in the photosynthetic cells tdy1 chlorotic sectors accumulate excess carbohydrates indicating that they have a defect in phloem loading . In agreement with this , we previously localized the site of TDY1 function to the innermost tissue layer of leaves comprised of the veins , bundle sheath cells , and interveinal mesophyll cells -LRB- Baker and Braun 2007 -RRB- . Due to the limits of the experiment it was not possible to further delineate where within this tissue layer TDY1 acts . However , the cytological investigations presented resolve this ambiguity . As we observed no blockages or alterations to plasmodesmatal structure along the sucrose symplastic pathway in tdy1-R leaves , the carbon accumulation points to a defect at a later step in phloem loading . Nonetheless , even though the plasmodesmata appear unobstructed , we can not be certain that they are functional . However , we propose that a molecular blockage of plasmodesmata precluding sucrose movement is an unlikely scenario for the following reason . In sxd1 mutants , specific plasmodesmata are occluded and unable to transit sucrose , causing the vascular parenchyma cells to appear plasmolysed due to their low osmotic concentration -LRB- Russin et al. 1996 -RRB- . Accordingly , we carefully inspected for , but never observed , plasmolysis of any vascular parenchyma , mesophyll or bundle sheath cells in tdy1 mutants in our microscopic analyses . Therefore , these data suggest that sucrose is able to pass freely between cells along the symplastic pathway , and hence that TDY1 likely functions in the veins -LRB- Fig. 6c -RRB- . From the variegated phenotype of tdy1 mutants , we hypothesize that TDY1 acts as a regulator of sucrose transport rather than as a transporter itself , as loss of transporter function leads to uniform carbohydrate accumulation and chlorosis phenotypes -LRB- Riesmeier et al. 1994 ; Burkle et al. 1998 ; Gottwald et al. 2000 ; see Braun et al. 2006 ; Baker and Braun 2007 for discussion of mechanisms for tdy1 variegation -RRB- . Several possibilities are envisioned for Tdy1 function . The first possibility is that Tdy1 may regulate sucrose efflux out of vascular parenchyma cells . Failure to export sucrose to the apoplast would create a bottleneck restriction leading to the build-up of sucrose in the vascular parenchyma cells and , in turn , to carbohydrate accumulation in the bundle sheath and mesophyll cells . The second possibility is that Tdy1 may function to regulate the activity of the H + - ATPases or sucrose transporters within the companion cells and sieve elements . Lack of function of either the H + - ATPases or sucrose transporters results in failure to load sucrose into the phloem and thereby to excess accumulation in the apoplast . Sucrose accumulation in the apoplast would shift the equilibrium for sucrose export from vascular parenchyma cells leading to increased concentration within these cells , and ultimately to carbon accumulation in the photosynthetic cells . In support of this , antisense expression or mutation of a phloem H + - ATPase or sucrose transporters causes an accumulation of carbohydrates in photosynthetic cells and chlorosis -LRB- Riesmeier et al. 1994 ; Burkle et al. 1998 ; Gottwald et al. 2000 ; Zhao et al. 2000 -RRB- . A third possibility is that TDY1 may function in the sucrose signal transduction pathway that regulates the abundance of the sucrose transporter controlling phloem loading -LRB- Chiou and Bush 1998 ; Vaughn et al. 2002 ; Ransom-Hodgkins et al. 2003 -RRB- . Our analyses of the tdy1-R mutant phenotype and genetic interactions between tdy1-R and sxd1-1 have shown that Tdy1 acts independently of Sxd1 . Thus , the tdy1 mutation identifies a separate genetic pathway limiting carbon accumulation in maize leaves . Future work characterizing the molecular function of Tdy1 will determine the mechanism by which Tdy1 influences carbon partitioning . Understanding the control of carbon export from leaves will lead to novel strategies to manipulate carbon allocation and biomass deposition which may have applications in the production of biofuels . Electronic supplementary material Below is the link to the electronic supplementary material . -LRB- DOC 31 kb -RRB-

O O B I I O O O O O O O B O O O O O O B I I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

carbohydrate accumulation
carbon partitioning
maize
sucrose export defective1


The atypical iron-coordination geometry of cytochrome f remains unchanged upon binding to plastocyanin , as inferred by XAS The transient complex between cytochrome f and plastocyanin from the cyanobacterium Nostoc sp. . PCC 7119 has been analysed by X-ray Absorption Spectroscopy in solution , using both proteins in their oxidized and reduced states . Fe K-edge data mainly shows that the atypical metal coordination geometry of cytochrome f , in which the N-terminal amino acid acts as an axial ligand of the heme group , remains unaltered upon binding to its redox partner , plastocyanin . This fact suggests that cytochrome f provides a stable binding site for plastocyanin and minimizes the reorganization energy required in the transient complex formation , which could facilitate the electron transfer between the two redox partners . Introduction In oxygen-evolving photosynthetic organisms , the cytochrome b6 -- f complex couples proton translocation across the thylacoid membrane to the electron transport between photosystems I and II -LRB- Allen 2004 -RRB- . In this complex , cytochrome f -LRB- Cf -RRB- transfers electrons from the Rieske iron sulphur cluster to a soluble metalloprotein that acts as the immediate electron donor of P700 cofactor at Photosystem I . The Cf subunit consists on a 28.2 kDa N-terminal soluble domain anchored to the membrane by C-terminal helix -LRB- Gray 1992 -RRB- . Its soluble domain is an atypical c-type cytochrome because both its β-sheet secondary structure and the unusual heme axial coordination -LRB- Martinez et al. 1994 -RRB- with the N-terminal amino acid Tyr-1 acting as an axial ligand . The most ubiquitous electron carrier between Cf and P700 is plastocyanin -LRB- Pc -RRB- -LRB- Sandman et al. 1983 -RRB- , which is a type I cupredoxin -LRB- Gough and Chotia 2004 -RRB- . Its structure consists on an anti-parallel β-barrel with a single copper atom -LRB- Coleman et al. 1978 ; Sykes 1985 ; Redinbo et al. 1994 -RRB- coordinated by two nitrogen atoms and two sulphur ones from highly conserved residues -LRB- H39 , C89 , H92 and M97 in Nostoc cyanobacterium -RRB- . The mechanism of the electron transfer -LRB- ET -RRB- reaction between Pc and its physiological partners has been studied extensively -LRB- Hope 2000 ; Hervás et al. 2003 ; Díaz-Quintana et al. 2003 -RRB- highlighting the role of electrostatic and hydrophobic interactions on binding . Also , the solution structures of several Pc -- Cf complexes have been reported -LRB- Ubbink 2004 ; Díaz-Moreno et al. 2005a , Lange et al. 2005 ; Musiani et al. 2005 -RRB- . In all the cases , these structures are consistent with a single conformation ensemble in which the hydrophobic patch surrounding Tyr-1 in Cf docks the hydrophobic patch of Pc . Despite the large amount of data concerning the nature of the interactions influencing the binding between the redox partners , little is known about the effects of complex formation on the metal cofactors , and how they can modulate the ET process . An analysis about whether the Fe coordination in Cf is altered upon binding to Pc would be interesting to address the above-mentioned effects , especially due to the fact that the N-terminal amino acid Tyr-1 , which is involved in the Pc -- Cf interface -LRB- Ubbink 2004 ; Díaz-Moreno et al. 2005a , Lange et al. 2005 ; Musiani et al. 2005 -RRB- , acts as axial ligand . To get a deep insight on these subjects we have studied the Fe K-edge of free and Pc-bound Cf by X-ray absorption spectroscopy -LRB- XAS -RRB- in solution . Materials and methods Protein samples Nostoc sp. . PCC 7119 Pc was purified from E. coli cells transformed with the pEAP-WT plasmid -LRB- Molina-Heredia et al. 1998 -RRB- . Production and purification of the soluble domain of Nostoc sp. . PCC 7119 Cf were as previously described -LRB- Albarrán et al. 2005 -RRB- . For the XAS experiments , the Pc and Cf samples were concentrated to the required volume by ultrafiltration methods , and exchanged into 10 mM sodium phosphate pH 6.0 . After concentration , stock solutions of 10 mM Pc and 4.5 mM Cf were obtained . Protein concentration was determined by optical spectroscopy using an absorption coefficient of 4.5 mM − 1 cm − 1 at 598 nm for oxidized Pc -LRB- Molina-Heredia et al. 1998 -RRB- and 31.5 mM − 1 cm − 1 at 556 nm for reduced Cf -LRB- Albarrán et al. 2005 -RRB- . Oxidized Cf -LRB- Cf III -RRB- was prepared by adding an equimolecular amount of potassium ferricyanide , followed by gel filtration in a Pharmacia Superdex G75 column to remove ferrocyanide . Reduced Cf -LRB- Cf II -RRB- was obtained upon addition of a tenfold excess of sodium ascorbate to a resulting Cf III sample . Oxidized Pc -LRB- PcII -RRB- was obtained by addition of either ferricyanide , as described for Cf. The oxidized complex -LRB- PcII -- Cf III -RRB- was prepared by adding an aliquot of a 10 mM PcII onto a Cf III sample up to reach a final concentration of ca. 2 mM each . Under these conditions , the percentage of Pc bound to Cf was estimated to be ca. 87 % , according to the binding constant calculated from NMR data -LRB- Díaz-Moreno et al. , 2005a -RRB- . The reduced complex -LRB- PcI -- Cf II -RRB- was obtained upon addition of a tenfold excess of sodium ascorbate to a PcII -- Cf III sample prepared as above . XAS measurements The X-ray absorption spectra were recorded at the European Synchrotron Radiation Facility -LRB- ESRF -RRB- in Grenoble -LRB- France -RRB- , which was operating with a ring current of 200 mA and energy of 6 GeV . The Fe K-edge -LRB- 7112 eV -RRB- was measured at beam line BM29 using a double crystal monochromator fitted with a pair of flat Si -LRB- 311 -RRB- crystals detuned to 50 % fwhm of the maximum transmission , for the suppression of high energy harmonics . All measurements were made at room temperature in fluorescence mode , using a 13-element Canberra solid-state germanium detector . The samples were contained in a PTFE cell equipped with two 12 μm Kapton foil windows . Energy calibration was achieved by measuring an iron foil for the Fe edge . The spectra of the foil were measured simultaneously , with the data in transmission mode , to be used as an internal reference . Ionization chambers filled with the appropriate mixture of gases were used as detectors . For all measurements , each data point was collected for 4 s , and several scans were averaged to achieve a good signal-to-noise ratio . In no case protein photoreduction or damage was observed , even in those samples in which the measurements were made over several hours . For the oxidized forms free and Pc-bound Cf , the edge remained at fix energy position from the first to the last scan , thus confirming the absence of protein photoreduction . The lack of radiation-induced changes in the samples was also confirmed by UV-vis measurements before and after XAS measurements . XAS data analysis The background subtraction required to obtain the extended X-ray absorption fine structure -LRB- EXAFS -RRB- functions χ -LRB- k -RRB- from the measured X-ray absorption spectra were performed using the AUTOBK code from the University of Washington . E0 was defined as the maximum of the first derivative of the absorption edge . To analyze the EXAFS spectra , the theoretical phases and amplitudes were calculated using the FEFF 6.0 code -LRB- Rehr et al. 1992 ; Newville et al. 1995 -RRB- . The fit to the experimental data was performed using the FEFFIT program -LRB- version 2.54 -RRB- -LRB- Newville et al. 1995 -RRB- . Results and discussion XANES region Figure 1 -LRB- upper panel -RRB- shows the X-ray absorption near edge structure -LRB- XANES -RRB- spectra corresponding to free Cf in its reduced and oxidized states . In both spectra there is a pre-edge feature corresponding to the forbidden transition 1s − 3d , which is sensitive to the electronic and geometric structure of the atom -LRB- Fe -RRB- site -LRB- Westre et al. 1997 -RRB- . In fact , the intensity of this transition agrees with the octahedral environment around the iron atom in Cf -LRB- Martinez et al. 1994 -RRB- . Fig. 1XANES region of the Fe K-edge XAS spectra of free and Pc-bound Cf. Upper , experimental data for oxidized and reduced forms of free proteins , Cf III -LRB- continuous line -RRB- and Cf II -LRB- dashed line -RRB- . Middle , data for the oxidized species , Cf III -LRB- continuous line -RRB- and PcII -- Cf III complex -LRB- dashed line -RRB- . Lower , data for the reduced species , Cf II -LRB- continuous line -RRB- and PcI -- Cf II complex -LRB- dashed line -RRB- . The presence of a pre-edge signal at 7113 eV is marked by arrows On the other hand , the edge position , determined by the maximum in the first derivative of the absorption spectrum corresponding to free Cf II , appears ca. 1 eV below that of Cf III , as previously found for the reduced and oxidized states of two closely related proteins , as heart cytochrome c -LRB- Cheng et al. 1999 -RRB- and Nostoc cytochrome c6 -LRB- Díaz-Moreno et al. 2006 -RRB- . In case of cytochrome c -LRB- Cheng et al. 1999 -RRB- , photoreduction of the oxidized species was observed only after the 16th spectrum of the series . In our case , however , there is no photoreduction as inferred from the fact that the edge position remains fixed for all the spectra along each series i.e. reduced or oxidized , free or bound . The reason not to observe any sample photo-reduction is due to the moderate brightness of the EXAFS spectrometer deliberately chosen for this investigation . In addition , the same shift has also been reported upon oxidation from Fe2 + to Fe3 + at pH 7 in the heme protein cytochrome c -LRB- Shulman et al. 1976 -RRB- . Moreover , there are some differences in both XANES spectra , particularly in the first feature after the edge . Figure 1 also shows the XANES regions of the absorption spectra of Cf bound to Pc in the oxidized and reduced state . The XANES region of the absorption spectra for PcII -- Cf III and Cf III are identical -LRB- Fig. 1 , middle panel -RRB- , indicating that the electronic and geometrical configuration around the iron centre remains unchanged when bound to Pc . However , this is not the case for the reduced proteins -LRB- Fig. 1 , lower panel -RRB- . In fact , the spectrum of the PcI -- Cf II complex exhibits some differences compared with that of free CfII . With regard to the pre-edge , a slight increase in the intensity of this feature is observed for CfII when bound to PcI , according to the decrease in electron density of the Fe atom . The shape of the first feature after the edge slightly changes too , thus suggesting certain modifications in the geometrical distribution of the ligands around the iron atom . It is worth noting that the spectrum of the reduced complex is very similar -- -- except for the pre-edge signal and edge position -- -- to that of the oxidized complex and free oxidized Cf , as shown in Fig. 3 . This suggests that the iron geometry in reduced Cf is distorted upon association to Pc , approaching to that of the iron centre in the oxidized form , either free or bound to Pc . EXAFS Region The extracted EXAFS signals of Cf in the reduced and oxidized states are shown in Fig. 2 -LRB- left panels -RRB- as k-weighted spectra . The choice of a k-weighting instead of a k3-weighting is based on the following facts . First , we were interested in determining the local environment around the absorbing atom and , in particular , at the first coordination shell . This shell is formed by iron and light elements , such as nitrogen , and their backscattering amplitude drops quickly at increasing energy -LRB- k value -RRB- . Therefore , they have a larger contribution when the transforms are made in k rather than in k3 . Second , if a k3-weighting is used , the highest energy part of the spectrum is emphasized and , in this case , the EXAFS signal would be dominated by the spectral region where the signal-to-noise ratio is poorer . Fig. 2EXAFS data -LRB- panels a and b -RRB- and their FT modules -LRB- panels c and d -RRB- at the Fe K-edge of Cf , either free or bound to Pc . The EXAFS spectra of free proteins -LRB- Cf II and CfIII -RRB- are shown in panela by continuous line for Cf II and dashed line for Cf III . In panel b , the EXAFS spectra of PcI -- Cf II and PcII -- Cf III complexes are represented by continuous and dashed lines , respectively . The corresponding FT modules of the EXAFS spectra for free Cf -LRB- Cf II and CfIII -RRB- are represented in panel c by closed and open circles , respectively . Panel d shows the FT modules for Pc-bound Cf forms : PcI -- Cf II by closed circles and PcII -- Cf III by open circles . The best fits of the FT data for both reduced -LRB- Cf II and PcI -- Cf II -RRB- and oxidized states -LRB- Cf III and PcII -- Cf III -RRB- are represented by continuous and dashed lines , respectively We should note here that the XAS data were collected at room temperature in order to maintain consistency with previous room temperature NMR measurements made on the same systems -LRB- Díaz-Moreno et al. 2005a , b -RRB- . Although a better signal to noise should be obtained at cryogenic temperatures , the transient complexes we study are not stable upon freezing , unless the partners are chemically cross-linked . Similar experimental conditions are required by EPR . Although EPR measurements on free and Pc-bound Cf would help to study the iron geometry , any comparison between results inferred from NMR or XAS , on one hand , and EPR , on the other , would be unreliable . As can be seen , the EXAFS function is not a simple wave but a combination of different waves corresponding to the contributions from the neighbouring atoms at different distances around the iron centre . Indeed , the EXAFS signals of Cf do not change upon binding to Pc , thereby indicating that the local coordination environment around the iron centre remains unchanged . The Fourier Transform -LRB- FT -RRB- of the corresponding spectra and the best fits , obtained with the parameters included in Table 1 , are also shown in Fig. 2 -LRB- right panels -RRB- . As a starting model , the values obtained from the X-ray diffraction -LRB- XRD -RRB- structure of Phormidium Cf -LRB- Carrel et al. 1999 -RRB- were used . In all cases there is a main signal around 1.5 Å , which corresponds to the first coordination sphere formed by the four nitrogen atoms of the porphyrin ring -LRB- NA , NB , NC and ND -RRB- , along with the nitrogen atoms of the two iron axial ligands Tyr-1 -LRB- N -RRB- and His-26 -LRB- Nε2 -RRB- . The intensities of the remaining signals are significantly lower than the first one because they correspond to contributions from lightweight elements lying at longer coordination distances . In order to fit the data , six simple scattering paths from the Fe atom to the six ligand N atoms were selected . Δr1 and Δr2 were used as two fitting parameters corresponding to changes in the distances of Fe to the two axial N atoms and to the four phorphyrin N atoms , respectively . The Debye -- Waller parameter , σ2 , related to system dynamic disorder was also fitted , with σ12 accounting for the bonds between the Fe atom and the two axial N atoms and σ22 accounting for the bonds between the Fe atom and the four porphyrin N atoms . The fifth adjustable parameter was the internal potential correction -LRB- ΔE0 -RRB- . Table 1Best-fit structural parameters resulted from the EXAFS analysis for free and Pc-bound Cf , in both oxidized and reduced stateSystemLigandΔE0 -LRB- eV -RRB- r -LRB- M -- L -RRB- -LRB- Å -RRB- σ2 -LRB- M -- L -RRB- -LRB- Å2 -RRB- CfIIIN -LRB- NC -RRB- − 7 ± 41.96 ± 0.020.002 ± 0.001 N -LRB- NA & ND -RRB- 1.97 ± 0.02 N -LRB- NB -RRB- 1.98 ± 0.02 N -LRB- Nε2 -RRB- 2.44 ± 0.060.010 ± 0.010 N -LRB- N -RRB- 2.49 ± 0.06 CfIIN -LRB- NC -RRB- − 6 ± 21.97 ± 0.010.002 ± 0.001 N -LRB- NA & ND -RRB- 1.99 ± 0.01 N -LRB- NB -RRB- 2.00 ± 0.01 N -LRB- Nε2 -RRB- 2.43 ± 0.020.004 ± 0.003 N -LRB- N -RRB- 2.48 ± 0.02 PcII -- CfIIIN -LRB- NC -RRB- − 7 ± 31.97 ± 0.010.002 ± 0.001 N -LRB- NA & ND -RRB- 1.98 ± 0.01 N -LRB- NB -RRB- 1.99 ± 0.01 N -LRB- Nε2 -RRB- 2.40 ± 0.030.011 ± 0.009 N -LRB- N -RRB- 2.45 ± 0.03 PcI -- CfIIN -LRB- NC -RRB- − 6 ± 31.97 ± 0.020.003 ± 0.001 N -LRB- NA & ND -RRB- 1.98 ± 0.02 N -LRB- NB -RRB- 1.99 ± 0.02 N -LRB- Nε2 -RRB- 2.40 ± 0.030.010 ± 0.009 N -LRB- N -RRB- 2.45 ± 0.03 S02 , factor of amplitude of reduction ; r -LRB- M -- L -RRB- , metal -- ligand distanceS02 = 1.0 , Δk = 3.5 -- 11 -LRB- 1/Å -RRB- , ΔR = 1.1 -- 2.1 -LRB- Å -RRB- , R factor -LRB- free Cf III -RRB- = 0.044 , R factor -LRB- free Cf II -RRB- = 0.004 , R factor -LRB- PcII-bound Cf III -RRB- = 0.004 , R factor -LRB- PcI-bound Cf II -RRB- = 0.003 Although there are no significant changes in the metal -- ligand distances , the Fe K-edge X-ray absorption data suggests a small distortion of the Fe2 + metal centre geometry when Cf II binds to PcI . The resulting Fe2 + geometry resembles that of Cf III , either free or bound to PcII -LRB- Fig. 3 -RRB- . Although the Fe atom of Cf shows a well-defined position at the heme group , which provides a rigid frame , the small distortion on the Fe2 + metal centre when Cf II binds to PcI can be explained by the smaller size of Fe3 + compared to Fe2 + : The Fe3 + atom fits well into the heme ring whereas the Fe2 + atom is somewhat shifted out of the plane -LRB- Schnackenberg et al. 1999 -RRB- ; however , the Fe2 + atom could be driven into the ring upon complex formation , thus yielding a structure of PcI-bound Fe2 + similar to that of Fe3 + in free and PcII-bound Cf III . Fig. 3XANES region of the Fe K-edge XAS spectra of free CfIII and PcI -- bound CfII . Experimental data for Cf III and PcI -- Cf II are shown in continuous and dashed lines . The presence of a pre-edge signal at 7113 eV is marked by arrows This tiny perturbation is consistent with the intensity changes at the pre-edge region of CfII in presence of PcI , because they correspond to transitions to the iron dz2 and dx2y2 orbitals , which are pointing towards the axial and equatorial ligands , respectively . As the reducing electron is not located in these orbitals , such subtle changes may have little influence on the redox potential . This situation is clearly different to that previously reported for cytochrome c6 , which shows a perturbation on the pre-edge region corresponding to its oxidized species , instead of the reduced ones , upon binding its reaction partner , photosystem I -LRB- Díaz-Moreno et al. 2006 -RRB- . In such case the pre-edge corresponds to transfer to a half-occupied non-bonding d orbital , which is that receiving the reducing electron , so the observed changes can be correlated with changes in the redox potential of the heme protein . Regarding to this point , it is important to note that the heme plane is normal to the interaction surface of cytochrome c6 , but parallel in the case of Cf. Thus , our results indicate that the bond between iron and the N-terminus nitrogen is strong enough to prevent a distortion of the iron-coordination geometry upon binding of Cf to Pc , as well as to provide a stable binding site to the copper protein . Such a stiffness of the coordination sphere minimises the reorganisation energy concomitant to charge transfer , thus optimising the ET process .

O O O O O B I O O O O O B O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

cytochrome f
electron transfer
plastocyanin
transient complexes

metalloproteins
x-ray absorption spectroscopy