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First published online September 29, 2006; 10.1105/tpc.105.039404 The Plant Cell 18:2710-2732 (2006) © 2006 American Society of Plant Biologists
Tocopherols Play a Crucial Role in Low-Temperature Adaptation and Phloem Loading in Arabidopsis[W]
a Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824 1 To whom correspondence should be addressed. E-mail dellapen{at}msu.edu; fax 517-353-9334.
To test whether tocopherols (vitamin E) are essential in the protection against oxidative stress in plants, a series of Arabidopsis thaliana vitamin E (vte) biosynthetic mutants that accumulate different types and levels of tocopherols and pathway intermediates were analyzed under abiotic stress. Surprisingly subtle differences were observed between the tocopherol-deficient vte2 mutant and the wild type during high-light, salinity, and drought stresses. However, vte2, and to a lesser extent vte1, exhibited dramatic phenotypes under low temperature (i.e., increased anthocyanin levels and reduced growth and seed production). That these changes were independent of light level and occurred in the absence of photoinhibition or lipid peroxidation suggests that the mechanisms involved are independent of tocopherol functions in photoprotection. Compared with the wild type, vte1 and vte2 had reduced rates of photoassimilate export as early as 6 h into low-temperature treatment, increased soluble sugar levels by 60 h, and increased starch and reduced photosynthetic electron transport rate by 14 d. The rapid reduction in photoassimilate export in vte2 coincides with callose deposition exclusively in phloem parenchyma transfer cell walls adjacent to the companion cell/sieve element complex. Together, these results indicate that tocopherols have a more limited role in photoprotection than previously assumed but play crucial roles in low-temperature adaptation and phloem loading.
Tocopherols are the best-studied class of lipid-soluble antioxidants and are produced only by photosynthetic organisms, including all plants and algae and some cyanobacteria. Structurally, all four tocopherols (  -, ß-,  -, and  -tocopherols) consist of a chromanol head group attached to a phytyl tail and differ only in the number and positions of methyl groups on the chromanol ring (Figure 1
). Tocopherols are amphipathic molecules, and in vitro studies using artificial membranes have shown that tocopherols form complexes with specific lipid constituents and physically stabilize membranes (Wassall et al., 1986 ; Stillwell et al., 1996; Wang and Quinn, 2000; Bradford et al., 2003). Tocopherols can efficiently quench singlet oxygen, scavenge various radicals, particularly lipid peroxy radicals, and thereby terminate lipid peroxidation chain reactions (Liebler and Burr, 1992; Bramley et al., 2000; Schneider, 2005). In animals, vitamin E deficiency results in muscular weakness and neurological dysfunction, which often coincide with increased lipid peroxidation (Machlin et al., 1977; Yokota et al., 2001). Recent studies have shown that tocopherols also have functions in animals unrelated to their antioxidant activity, such as modulation of cell signaling and transcriptional regulation (Ricciarelli et al., 1998; Jiang et al., 2000; Rimbach et al., 2002; Kempna et al., 2004). In contrast with the extensive studies of tocopherol functions in animals, we are only beginning to understand tocopherol functions in the photosynthetic organisms in which they are produced. In plants, tocopherols are synthesized and localized in plastid membranes that are also highly enriched in polyunsaturated fatty acids (PUFAs) (Bucke, 1968; Soll et al., 1980, 1985; Lichtenthaler et al., 1981; Soll, 1987; Vidi et al., 2006), and increased tocopherol content has been correlated in the response of photosynthetic tissues to a variety of abiotic stresses, including high-intensity light (HL), salinity, drought, and low temperatures (Munne-Bosch et al., 1999; Keles and Oncel, 2002; Bergmuller et al., 2003; Collakova and DellaPenna, 2003).
Such data, together with the evolutionary conservation of tocopherol synthesis among photosynthetic organisms, has led to the assumption that a primary function of tocopherols is to protect photosynthetic membranes from oxidative stresses by acting as lipid-soluble antioxidants (Fryer, 1992 ; Munne-Bosch and Alegre, 2002). Although plausible, such hypotheses are based primarily on correlations and circumstantial evidence and have yet to be rigorously tested in planta. The isolation of Arabidopsis thaliana mutants disrupting steps of the tocopherol biosynthetic pathway provides powerful tools with which to directly investigate tocopherol functions in plants (Figure 1) (Shintani and DellaPenna, 1998; Collakova and DellaPenna, 2001; Porfirova et al., 2002; Cheng et al., 2003; Sattler et al., 2003; DellaPenna and Pogson, 2006). The vitamin E2 (vte2) mutant is defective in homogentisate phytyl transferase (HPT) and lacks all tocopherols and pathway intermediates (Figure 1, Table 1
). vte2 mutants are severely impaired in seed longevity and early seedling development as a result of the massive and uncontrolled peroxidation of storage lipids (Sattler et al., 2004; S.E. Sattler, L. Mene-Saffrane, E.E. Farmer, M. Krischke, M.J. Mueller, and D. DellaPenna, unpublished data), consistent with the loss of the lipid-soluble antioxidant functions of tocopherols (Ham and Liebler, 1995, 1997). Interestingly, the vte2 mutants that do survive early seedling development become virtually indistinguishable from wild-type plants under standard growth conditions (Sattler et al., 2004; this study), suggesting that unlike seed longevity and germination, tocopherols are dispensable in mature plants in the absence of stress. Consistent with this, constitutive overexpression of VTE2 in Arabidopsis increased total leaf tocopherols by 4.5-fold but had no discernible effect relative to the wild type on plant growth or chlorophyll and carotenoid content in the absence of stress or under combined nutrient and HL stresses (Collakova and DellaPenna, 2003).
The vte1 mutant is defective in tocopherol cyclase activity and deficient in all tocopherols, but unlike vte2, it accumulates the redox-active biosynthetic intermediate 2,3-dimethyl-6-phytyl-1,4-benzoquinol (DMPBQ) (Figure 1, Table 1) (Sattler et al., 2003 ). When grown at 100 to 120 µmol·m–2·s–1, vte1 plants are virtually identical to wild-type plants at all developmental stages (Porfirova et al., 2002; Sattler et al., 2003, 2004). The lipid-peroxidation phenotype observed in germinating vte2 seedlings was not observed in vte1, indicating that DMPBQ can fully compensate for tocopherols as a lipid-soluble antioxidant in seedlings (Sattler et al., 2004). Under HL stress (5 d at 850 µmol·m–2·s–1 [Porfirova et al., 2002]) or a combination of low-temperature and HL stresses (5 d at 6 to 8°C and 1100 µmol·m–2·s–1 [Havaux et al., 2005]), vte1 was nearly identical to the wild type for all parameters measured, including lipid peroxidation, with the exception of a slight decrease in maximum photosynthetic efficiency (Fv/Fm). Only under extreme conditions (24 h at 3°C and continuous 1500 to 1600 µmol·m–2·s–1) did vte1 show a more rapid induction of lipid peroxidation than the wild type, although this difference was transient, and after 48 h of treatment lipid peroxidation was increased similarly in vte1 and wild-type plants (Havaux et al., 2005). These studies with vte1 and vte2 mutants suggest that a primary function of tocopherols is to control nonenzymatic lipid oxidation, especially during seed storage and early germination, and also probably in photosynthetic tissues, but only under the most extreme of combined HL and low-temperature stresses.
Interestingly, a maize (Zea mays) tocopherol cyclase mutant (sucrose export defective1 [sxd1]) was identified several years before the identification of Arabidopsis vte1, not because of its impact on tocopherol synthesis but because of the accumulation of carbohydrates and anthocyanins in sxd1 source leaves, which coincided with aberrant plasmodesmata between the bundle sheath and vascular parenchyma cells (Russin et al., 1996 In this study, we further define and clarify the physiological role(s) of tocopherols in photosynthetic plant tissues by subjecting and analyzing the response of a suite of Arabidopsis tocopherol mutants to a variety of abiotic stresses. We report that in contrast with long-held assumptions about tocopherol functions in plants, tocopherol-deficient mutants are remarkably similar to wild-type plants in their response to most abiotic stresses, with the notable exception of an increased sensitivity to nonfreezing low temperatures. Detailed physiological, biochemical, and ultrastructural data demonstrate that the earliest impact of tocopherol deficiency during low-temperature treatment is an inhibition of photoassimilate transport associated with dramatic structural changes in phloem parenchyma transfer cells, a bottleneck for photoassimilate transport. The resulting accumulation of carbohydrates in source leaves affects the physiology and response of the entire plant to low temperatures.
Tocopherol Biosynthetic (vte) Mutants Used in This Study vte1-1, vte1-2, and vte2-1 are previously isolated and characterized ethyl methanesulfonate mutants in the Columbia (Col) ecotype that are deficient in the tocopherol cyclase (vte1-1 and vte1-2) and HPT (vte2-1) enzymes (Sattler et al., 2003 , 2004) (Figure 1). vte2-2 and vte4-3 are T-DNA insertion mutants in the Wassilewskija (Ws) ecotype in genes encoding HPT and -tocopherol methyltransferase, respectively. Leaves of all mutants, vte2-1, vte2-2, vte1-1, vte1-2, and vte4-3, lack -tocopherol, the major tocopherol in wild-type Arabidopsis leaves (Figure 1, Table 1) (Sattler et al., 2003). vte2-1 and vte2-2 lack all tocopherols and pathway intermediates. vte1-1 and vte1-2 lack all tocopherols but accumulate the biosynthetic pathway intermediate DMPBQ at a level comparable to -tocopherol in Col. The vte4-3 mutant accumulates -tocopherol at an equivalent or slightly higher level than -tocopherol in Ws. Three- to 5-week-old plants of all mutant genotypes grown under permissive conditions (12 h of 120 µmol·m–2·s–1 light at 22°C and 12 h of darkness at 18°C) were virtually identical to their respective wild-type backgrounds, consistent with previous reports that mutations disrupting tocopherol synthesis have little impact on the normal growth of mature plants (Porfirova et al., 2002; Bergmuller et al., 2003; Sattler et al., 2003, 2004).
Response of Tocopherol-Deficient Mutants to HL Stress
To assess changes in photosynthetic pigment and tocopherol levels in response to HL stress, the seventh to ninth oldest leaves were harvested before and after 4 d of HL1800 for HPLC analysis. Before HL1800, all levels were similar between genotypes except for the slightly lower ß-carotene content in vte2-1 and vte1-1 relative to Col and the absence of tocopherols in all vte genotypes (see Supplemental Table 1 online). After 4 d of HL1800, the vte mutants generally had lower total and individual chlorophyll levels than Col, but these differences were not significant in all cases after 4 d of HL1800, even with n = 19 (Figure 2B, Table 2 ; see Supplemental Figure 2 online). Total carotenoids were consistently and significantly lower in Col than in vte1-1 and vte1-2, but not always in vte2-1, after 4 d of HL1800. Neoxanthin and violaxanthin were significantly lower in all vte mutants, whereas lutein was significantly lower only in vte1-1 and vte1-2. Interestingly, zeaxanthin was 70% higher in Col than in vte2-1 but unchanged relative to Col in both vte1 alleles (Table 2).
In vivo chlorophyll a fluorescence was also analyzed to assess photosystem II (PSII) function during HL stress. Typically, when plants are under oxidative stress, PSII is inactivated as a result of enhanced turnover of the D1 protein, a process termed photoinhibition, and Fv/Fm decreases (Bjorkman and Demmig, 1987 ; Maxwell and Johnson, 2000). Four-week-old Col, vte2-1, vte1-1, and vte1-2 plants grown under permissive conditions had identical Fv/Fm values of between 0.8 and 0.85, typical values for healthy leaves (Bjorkman and Demmig, 1987; Maxwell and Johnson, 2000) (data not shown). After 24 h of HL1800 (8 h of HL1800, 8 h of darkness, and 8 h of HL1800), a few vte2-1 leaves showed a dramatic reduction in Fv/Fm (<0.5), but the majority had values similar to Col, and the average Fv/Fm of vte2-1 was not significantly different from Col, even with n = 30 (Figure 2D; see Supplemental Figure 2 online). By contrast, vte1-1 and vte1-2 both had more leaves with Fv/Fm < 0.5 and average Fv/Fm values that were significantly lower than Col (Figure 2D; see Supplemental Figure 2 online). These combined results indicate that the elimination of tocopherols in vte2 has surprisingly little impact on the response of the photosynthetic apparatus to HL stress in comparison with Col, with the exception of altered xanthophyll cycle carotenoids. Equally surprising is the fact that although the vte2 and vte1 genotypes are identical with regard to their tocopherol deficiencies, vte1 alleles are slightly more susceptible to HL1800 than vte2. As the primary biochemical difference between these two genotypes is that vte1 mutants accumulate the redox-active intermediate DMPBQ and vte2 mutants do not, the presence of DMPBQ in vte1 may have negative effects on HL stress tolerance in Arabidopsis.
Tocopherol-Deficient Mutants Exhibit a Low-Temperature-Sensitive Phenotype
After transfer to 7.5°C, vte2-1 and Col did not differ in time to bolting (53 ± 4 and 51 ± 3 d, respectively) or in the number of leaves produced at the start of bolting (32 ± 3 and 31 ± 2 leaves, respectively), indicating that the process of vernalization was not affected by the lack of tocopherols. However, after prolonged growth at 7.5°C, vte2-1 siliques were shorter, produced significantly fewer seeds per silique and per plant compared with Col, and 35% of the seeds in vte2-1 siliques were aborted compared with <1% in Col siliques (Figure 3E, Table 3 ). These results indicate that tocopherols play a crucial role in low-temperature adaptation in Arabidopsis.
Subjecting vte1-1 to 7.5°C treatment resulted in a phenotype intermediate between vte2-1 and Col in terms of overall growth, mature leaf color, silique size, number of seeds per silique, percentage of aborted seeds, and seed yield per plant (Figure 3, Table 3). These phenotypes in 7.5°C-treated vte4-3 were virtually indistinguishable from those in wild type Ws (Figures 3A to 3C). These results indicate that during low-temperature adaptation in Arabidopsis, the quinol biosynthetic intermediate DMPBQ partially compensates for the lack of tocopherols in vte1-1, whereas the -tocopherol accumulated in vte4-3 leaves can functionally replace -tocopherol in this regard.
Photooxidative Damage Is Not Associated with the vte2 Low-Temperature Phenotype
Consistent with the purple color of mature leaves of 7.5°C-treated vte2 mutants (Figure 3B), vte2-1 accumulated significantly higher levels of anthocyanins than Col after 14 d of 7.5°C (Figure 4D). In Col, anthocyanins were detected only at 7 d. Because anthocyanin accumulation is often associated with plant responses to stress (Leyva et al., 1995 ; Chalker-Scott, 1999) and tocopherols are well-characterized lipid-soluble antioxidants in animals (Ham and Liebler, 1995, 1997), it seemed plausible that increased lipid peroxidation might be occurring in vte2-1 during low-temperature treatment. However, the lipid peroxide levels of vte2-1 and Col analyzed by the ferrous oxidation xylenol orange assay were found to be similar and near background levels at all time points (Figure 4B), indicating that the observed phenotypic differences between 7.5°C-treated vte2-1 and Col are not associated with a detectable increase in lipid peroxidation.
Given the reported localization of tocopherols and tocopherol biosynthetic enzymes to plastids (Bucke, 1968
To assess the response of the photosynthetic apparatus to 7.5°C, changes in photosynthetic parameters were analyzed. Fv/Fm was unchanged in both Col and vte2-1 at any time point (Figure 5A
), indicating that photoinhibition is not occurring in either genotype during permissive or 7.5°C conditions. The quantum yield of PSII (
Tocopherol-Deficient Mutants Accumulate Carbohydrates during Low-Temperature Treatment The reduced  PSII in vte2-1 after 14 d could result from feedback inhibition of photosynthesis attributable to the accumulation of downstream carbon metabolites (Goldschmidt and Huber, 1992; Koch, 1996; Paul and Foyer, 2001; Paul and Peliny, 2003). To assess this possibility, starch, glucose, fructose, and sucrose contents were analyzed during the time course of 7.5°C treatment. Starch represents the main plastidic carbohydrate storage pool, sucrose and fructose are cytosolic pools, and glucose is present in both subcellular compartments. Col and vte2-1 had identical carbohydrate contents at the end of the light period under permissive growth conditions (Figure 6
, day 0). During the first 7 d of 7.5°C treatment, starch content increased similarly in both Col and vte2-1 to  120 µmol glucose equivalents/g fresh weight. After 7 d, vte2-1 starch content increased steadily to 680 µmol glucose equivalents/g fresh weight, whereas Col starch levels decreased to near initial levels (Figure 6A). Likewise, the glucose, fructose, and sucrose contents of Col and vte2-1 increased similarly during the first 3 d of low-temperature treatment (Figures 6B to 6D), likely as a component of the well-documented cold acclimation response(s) in Arabidopsis (Wanner and Junttila, 1999; Taji et al., 2002). After 3 d, Col soluble sugar levels decreased, whereas those in vte2-1 continued to increase, reaching 35, 43, and 255 times the initial levels of glucose, fructose, and sucrose, respectively, after 28 d of low-temperature treatment. The timing of the increase and accumulation of carbohydrates in vte2-1 is consistent with this being the root cause of the reduction in PSII observed after 14 d at 7.5°C (Figure 5B).
To further investigate any differences in carbohydrate accumulation between vte2-1 and Col during the initial 5 d of low-temperature treatment, diurnal changes in carbohydrate contents were analyzed 1 h before the end of the light and dark cycles. During the 25 h before low-temperature treatment (Figure 7 , –25, –13, and –1 h, with 0 h being the transfer time of plants to low temperature at the start of the light cycle), starch, glucose, fructose, and sucrose contents were almost identical in vte2-1and Col. These data indicate that the lack of tocopherols does not have a significant impact on carbohydrate metabolism under permissive growth conditions. After transfer to low temperature, the soluble sugar content increased similarly in vte2-1 and Col for the first two diurnal cycles, with significant differences first being observed between genotypes at the end of the third low-temperature light period (Figures 7B to 7D, 59 h). By contrast, starch levels did not become significantly different between genotypes until 14 d of low-temperature treatment (Figures 6A and 7A). The differential increase of soluble sugars before starch accumulation in vte2-1 indicates that the increase in cytosolic soluble sugars precedes starch accumulation in the chloroplast and that soluble sugars are not being efficiently metabolized or mobilized in 7.5°C-treated vte2-1.
The vte2 and vte1 Cold-Sensitive Phenotypes Are Attenuated in Young Leaves Mature (7th to 9th oldest) and young (13th to 16th oldest) leaves of vte2-1 and vte1-1 mutants showed obvious visible differences in their responses to low temperature; young leaves of vte2-1 and vte1-1 did not change their color to purple even after 2 months of 7.5°C treatment (Figure 3C). Consistent with these visual observations, mature leaves of vte1-1 had an anthocyanin content 10% that of vte2-1 but still higher than that of Col, whereas young vte2-1 and vte1-1 leaves accumulated many fewer anthocyanins compared with their respective mature leaves after 28 d of 7.5°C treatment (Figure 8A ). Fv/Fm was >0.8 in all cases, indicating that photoinhibition was not occurring in either young or mature leaves of any genotype (data not shown). The PSII of mature vte2-1 leaves was reduced to 70% of that of mature Col leaves, consistent with Figure 5, whereas the PSII of mature vte1-1 leaves was only slightly decreased relative to mature Col leaves. However, the PSII of mature and young Col leaves and young vte2-1 and vte1-1 leaves was not significantly different (Figure 8B). Levels of all carbohydrates in mature vte2-1 leaves were greatly increased compared with Col, consistent with Figure 6. Mature vte1-1 leaves contained intermediate levels of starch, glucose, fructose, and sucrose (51, 53, 68, and 58% of vte2-1 levels, respectively) (Figures 8C to 8F). Young vte2-1 and vte1-1 leaves contained substantially reduced starch, glucose, and sucrose levels compared with their respective mature leaves. These results indicate that the initiation and development of young vte2-1 and vte1-1 leaves under 7.5°C conditions attenuates the biochemical phenotypes observed in mature leaves of both genotypes and that the DMPBQ accumulated in vte1-1 further suppresses these biochemical phenotypes in both mature and young leaves.
Tocopherol-Deficient Mutants Have Reduced Source Leaf Photoassimilate Export Capacity at Low Temperatures The reduced seed yield (Table 3) and attenuated carbohydrate accumulation in young leaves relative to mature leaves (Figure 8) in 7.5°C-treated vte2-1 suggested an impaired translocation of photoassimilates from mature source tissues to young sink tissues. To test this possibility, 14CO2 labeling experiments were conducted. Col and vte2-1 were grown on plates under permissive conditions for 3 weeks and then transferred to 7.5°C for an additional 7 d. Whole plants were labeled with 14CO2 at 7.5°C, transferred to high humidity in darkness at 7.5°C for 2 h to allow for photoassimilate transport, and subsequently exposed to a phosphor screen to visualize the movement of 14C-labeled photoassimilate. Immediately after labeling, >99% of the 14CO2 incorporated was present in leaf tissue (data not shown). Col and vte2-1 incorporated similar amounts of 14CO2 into photosynthate, suggesting that their carbon fixation rates do not differ, consistent with the similar PSII within the first 7 d at 7.5°C (Figure 5B; data not shown). After the 2-h dark period, Col had translocated 13.2% of the 14C-labeled photoassimilate fixed in leaves to roots, whereas only 2.7% was translocated in vte2-1 (Figure 9A
). These results demonstrate that vte2-1 translocates significantly less photoassimilate from source to sink than Col after 7 d of 7.5°C treatment.
Impaired photoassimilate translocation in 7.5°C-treated vte2-1 could be attributable to reduced sink strength or impaired photoassimilate export from source leaves (Stitt, 1996 ; Herbers and Sonnewald, 1998; Gottwald et al., 2000). To address these possibilities, phloem exudation experiments were conducted (King and Zeevaart, 1974). Col and vte2-1 were grown for 4 weeks under permissive conditions and transferred to 7.5°C for an additional 0, 1, 3, or 7 d. Mature (seventh to ninth oldest) leaves were excised from plants and labeled with 14CO2. The petioles of labeled leaves were then transferred to an EDTA solution to induce phloem exudation, and radioactivity in the EDTA solution was determined at various time points (King and Zeevaart, 1974). Again, total 14CO2 fixed in mature leaves was similar in all genotypes at each time point (Figure 9C). Before 7.5°C treatment (day 0), Col, vte1-1, and vte2-1 leaves exuded similar amounts of labeled photoassimilates, accounting for 34% of the total 14CO2 fixed in each genotype. During 7.5°C treatment, the percentage exudation by Col decreased slightly after 3 and 7 d (to 27 and 31% of the total 14CO2 fixed, respectively), whereas that of vte2-1 was greatly reduced (to 11 and 4% at 3 and 7 d, respectively). Even more intriguingly, exudation in vte2-1 was significantly lower than that in Col during the first day of 7.5°C treatment, which corresponds to only 6 h of 7.5°C treatment. The vte1-1 mutant exuded 17 and 15% of the total 14CO2 fixed after 3 and 7 d at 7.5°C, respectively, levels intermediate between Col and vte2-1 (Figure 9C).
In apoplastic loaders such as Arabidopsis, sucrose is almost the exclusive translocated photoassimilate (Vanbel, 1993 Overall, the results obtained from 14CO2 labeling experiments indicate that tocopherol deficiency in both vte1-1 and vte2-1 results in a dramatically reduced capacity of photoassimilate export from source leaves in response to 7.5°C treatment. The rapidity of the reduction in photoassimilate export in 7.5°C-treated vte2-1 strongly suggests that impairment of photoassimilate export is the root cause of the sugar accumulation phenotype observed in mature leaves of 7.5°C-treated tocopherol-deficient mutants.
Structural Changes in Low-Temperature-Treated Tocopherol-Deficient Mutants
To confirm whether or not aniline blue–positive fluorescence was cell-specific and could be attributed to callose deposition, serial sections of 0- and 14-d 7.5°C-treated Col and vte2-1 vascular tissue were examined at the level of the transmission electron microscope. The spatial organization of cells and types of cells constituting the phloem and xylem of both Col and vte2-1 were identical to what has been described previously for Arabidopsis (Haritatos et al., 2000 ) (data not shown). Notably, at day 0, phloem vascular parenchyma cells of both Col and vte2-1 contained transfer cell wall ingrowths adjacent to sieve elements and companion cells (Figure 12A
). Treatment of Col at 7.5°C for 14 d did not result in obvious ultrastructural changes in any vascular cell type except for a noticeable increase in phloem parenchyma transfer cell differentiation and transfer cell wall deposition exclusively adjacent to sieve elements and companion cells in all vascular tissues (Figure 12B), although to a lesser degree in the midvein.
During the same time course of 7.5°C treatment in vte2-1, changes in cell fine structure occurred exclusively within the phloem parenchyma transfer cells of all vascular traces. Phloem parenchyma transfer cells in 14-d-treated vte2-1 exhibited irregularly thickened cell wall depositions with ultrastructural features characteristic of callose (Nishimura et al., 2003 ) (Figures 12C to 12F). Large callose-like masses that dissected the cell lumen corresponded in shape to aniline blue–positive fluorescent regions (cf. Figures 12C and 12E with Figures 10F, 10I, and 10L). The callose-like wall material also formed a sheath around the cells (Figure 12D) and was deposited over transfer cell wall ingrowths (Figure 12F) and between the end walls of adjoining transfer cells, including plasmodesmata (data not shown). Immunolocalization using monoclonal antibodies against callose confirmed the presence of callose at each location (Figures 12G to 12J) and at plasmodesmata between the phloem parenchyma transfer cells and the bundle sheath (Figure 12K). No immunolabeling was present in controls using secondary antibody only (Figures 12D to 12F), and immunolabeling was rare to absent in all cell types of untreated Col and vte2-1 and in 14-d, 7.5°C-treated Col, including phloem parenchyma transfer cells (Figure 12L). Serial sections of vascular tissue from Col and vte2-1 treated at 7.5°C for 3 and 7 d were subsequently examined at the level of the transmission electron microscope to determine the spatial and temporal development of callose deposition within phloem parenchyma cells. At 3 d, phloem parenchyma transfer cell wall deposition in Col was confined to the sieve element or companion cell boundary (data not shown), but in vte2-1, wall deposition was present around the entire transfer cell periphery (Figure 13A ). Cell wall deposition in 3-d-treated vte2-1 resulted in abnormally thickened and irregular shaped ingrowths with callose-like depositions adjacent to sieve elements and companion cells (Figure 13A) that grew increasingly prominent by day 7 (data not shown). In 3-d, 7.5°C-treated vte2-1, positive immunolocalization with monoclonal antibodies to callose was present exclusively at the phloem parenchyma transfer cell wall/sieve element boundary (Figure 13B) and included phloem parenchyma transfer cell–sieve element plasmodesmata connections (Figure 13C). In contrast with 14 d, plasmodesmata between bundle sheath and phloem vascular parenchyma cells in 3-d, 7.5°C-treated vte2-1 were continuous and immunonegative for callose (cf. Figures 12K and 13D).
The chemistry of tocopherols as lipid-soluble antioxidants and terminators of PUFA free radical chain reactions has been well established from analyses in artificial membranes and animal-derived membrane systems (Liebler and Burr, 1992 ; Ham and Liebler, 1995). It has long been assumed that similar chemistry occurs in the tocopherol-containing PUFA-enriched membranes of photosynthetic organisms, and this assumption has recently been supported by studies of tocopherol-deficient photosynthetic organisms (Sattler et al., 2004; Havaux et al., 2005; Maeda et al., 2005). During the first several days of germination, a period of high oxidative metabolism, Arabidopsis vte2 seedlings contain levels of oxidized lipids >100-fold higher than wild-type or vte1 seedlings, which were indistinguishable (Sattler et al., 2004; S.E. Sattler, L. Mene-Saffrane, E.E. Farmer, M. Krischke, M.J. Mueller, and D. DellaPenna, unpublished data). Thus, a chemical role for tocopherols (or DMPBQ in vte1) in limiting lipid peroxidation appears to be conserved between photosynthetic organisms and animals. By 18 d of growth, the lipid peroxide levels of vte2 seedlings had decreased to near wild-type and vte1 levels (Sattler et al., 2004), and they became indistinguishable from those of the wild type and vte1 after 4 weeks of growth (Figure 4B), consistent with tocopherols being dispensable in mature photosynthetic tissues in the absence of stress (Porfirova et al., 2002; Sattler et al., 2003, 2004). Although a chemical role for tocopherols in controlling lipid peroxidation at specific points of the plant life cycle now seems clear, the physiological roles of tocopherols during plant stresses do not. In this study, we used a suite of Arabidopsis vte mutants that accumulate different types and levels of tocopherols and pathway intermediates (Table 1) to directly assess tocopherol-specific functions in photosynthetic tissues in planta in response to abiotic stress treatments.
A Limited Role for Tocopherols in Protecting Arabidopsis Plants from HL Stress
One possible explanation for this surprisingly limited role of tocopherols during HL stress is that other mechanisms compensate for their absence. The zeaxanthin level of HL1800 vte2 was nearly twice that of Col (Table 2). vte2 (and vte1) also had a higher xanthophyll deepoxidation state (A+Z/A+Z+V) after HL1800 treatment (see Table 2 for definitions), as did vte1 during HL stress combined with low or high temperatures (Havaux et al., 2005
In previous studies, it was demonstrated that, although vte1 and vte2 are both tocopherol-deficient, the two genotypes behave quite differently during early seedling development: vte2 exhibited a >100 fold increase in nonenzymatic lipid peroxidation during germination, whereas lipid peroxidation in vte1 was identical to that in the wild type (Sattler et al., 2004
Arabidopsis Tocopherol-Deficient Mutants Exhibit a Cold-Sensitive Phenotype Independent of Photooxidative Damage
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ABSTRACT
INTRODUCTION
