FIBRILLIN4 is required for plastoglobule development and stress resistance in apple and Arabidopsis.

The fibrillins are a large family of chloroplast proteins that have been linked with stress tolerance and disease resistance. FIBRILLIN4 (FIB4) is found associated with the photosystem II light-harvesting complex, thylakoids, and plastoglobules, which are chloroplast compartments rich in lipophilic antioxidants. For this study, FIB4 expression was knocked down in apple (Malus 3 domestica) using RNA interference. Plastoglobule osmiophilicity was decreased in fib4 knockdown (fib4 KD) tree chloroplasts compared with the wild type, while total plastoglobule number was unchanged. Compared with the wild type, net photosynthetic CO2 fixation in fib4 KD trees was decreased at high light intensity but was increased at low light intensity. Furthermore, fib4 KD trees produced more anthocyanins than the wild type when transferred from low to high light intensity, indicating greater sensitivity to high light stress. Relative to the wild type, fib4 KD apples were more sensitive to methyl viologen and had higher superoxide levels during methyl viologen treatment. Arabidopsis (Arabidopsis thaliana) fib4 mutants and fib4 KD apples were more susceptible than their wild-type counterparts to the bacterial pathogens Pseudomonas syringae pathovar tomato and Erwinia amylovora, respectively, and were more sensitive to ozone-induced tissue damage. Following ozone stress, plastoglobule osmiophilicity decreased in wild-type apple and remained low in fib4 KD trees; total plastoglobule number increased in fib4 KD apples but not in the wild type. These results indicate that FIB4 is required for plastoglobule development and resistance to multiple stresses. This study suggests that FIB4 is involved in regulating plastoglobule content and that defective regulation of plastoglobule content leads to broad stress sensitivity and altered photosynthetic activity.

Increased production of reactive oxygen species (ROS) is among the first biochemical responses of plants when challenged by pathogens and harsh environmental conditions (Mehdy, 1994;Lamb and Dixon, 1997;Joo et al., 2005). ROS are implicated in tissue damage during environmental stress and in the promotion of disease development by necrotrophic and hemibiotrophic pathogens (Venisse et al., 2001;Apel and Hirt, 2004;Shetty et al., 2008). For example, ROS production is critical for host colonization and pathogenesis by the bacterium Erwinia amylovora, which causes fire blight disease in rosaceous plants such as apple (Malus 3 domestica) and pear (Pyrus communis; Venisse et al., 2001).
The chloroplast is a site of ROS production during biotic and abiotic stress (Joo et al., 2005;Liu et al., 2007). The chloroplast has a battery of enzymes such as superoxide dismutase and ascorbate peroxidase, and antioxidants such as ascorbate, glutathione, and tocopherols, for protection against ROS (Noctor and Foyer, 1998;Asada, 2006). Plastoglobules are lipoprotein bodies attached to the thylakoids  that store lipids, including antioxidants such as tocopherols, carotenes, and plastoquinones (Steinmü ller Tevini and Steinmü ller, 1985). In addition to antioxidants, plastoglobules contain tocopherol cyclase, which is involved in g-tocopherol synthesis Vidi et al., 2006). The antioxidant content of plastoglobules and their apparent involvement in tocopherol biosynthesis imply that they could play a role in plant responses to oxidative stress.
The Arabidopsis fibrillin encoded by At3g23400 has received various appellations, including FIBRILLIN4 (FIB4; Laizet et al., 2004), Harpin-Binding Protein1 (Song et al., 2002), AtPGL 30.4 , and Fibrillin6 (Galetskiy et al., 2008); here, it will be referred to by its earliest published name, FIB4. FIB4 is found associated with the PSII light-harvesting complex (Galetskiy et al., 2008). FIB4 has also been detected in plastoglobules Ytterberg et al., 2006) and thylakoids (Friso et al., 2004;Peltier et al., 2004). However, the specific function of FIB4 is unknown. Several lines of evidence suggest that FIB4 may be involved in plant disease resistance responses: pathogen-associated molecular patterns trigger its phosphorylation (Jones et al., 2006); pathogen-associated molecular patterns stimulate the expression of its ortholog in tobacco (Nicotiana tabacum; Jones et al., 2006;Sanabria and Dubery, 2006); and it can physically interact with the HrpN (harpin) virulence protein of the fire blight pathogen E. amylovora in a yeast twohybrid assay, suggesting that it could be a receptor or target of HrpN (Song et al., 2002). In addition, it is thought that FIB4 may be involved in the transport of small, hydrophobic molecules because it contains a conserved lipocalin signature (Jones et al., 2006). Here, we report a genetic analysis of FIB4 function in apple and Arabidopsis in terms of its role in plastoglobule development and plant resistance to biotic and abiotic stresses.

Knockdown of FIB4 Expression in Apple
We generated fib4 knockdown (fib4 KD) apple trees using the RNA interference (RNAi) technique in the cv Royal Gala (Supplemental Figs. S1 and S2A; Maximova et al., 1998). Transgenic, vector-only control (VC) plants carrying the transformation marker genes Neomycin Phosphotransferase II and Enhanced Green Fluorescent Protein (EGFP), but not the FIB4 RNAi cassette, were also generated (Supplemental Figs. S1 and S2A). The level of FIB4 transcript in fib4 KD plantlets was less than 10% of the wild-type level, indicating suc-cessful silencing (Supplemental Fig. S2B). Wild-type, VC, and fib4 KD apple trees were phenotypically indistinguishable when grown under normal laboratory conditions (Supplemental Fig. S2C).

Plastoglobule Ultrastructure
Since FIB4 is a component of the plastoglobule and is associated with the PSII light-harvesting complex, we investigated the effect of knockdown of FIB4 expression on chloroplast and plastoglobule ultrastructure in apple. Leaf sections from fib4 KD and wild-type apple trees were stained with osmium tetroxide (OsO 4 ) and observed by transmission electron microscopy (TEM). Overall chloroplast morphology appeared normal in fib4 KD palisade mesophyll cells (Fig. 1A). Strikingly, however, fib4 KD chloroplasts contained almost exclusively electron-transparent (nonosmiophilic, or white) plastoglobules, while wildtype chloroplasts contained many electron-opaque (osmiophilic, or black) plastoglobules (Fig. 1, A and B). Plastoglobules are thought to be osmiophilic because the unsaturated lipids they contain reduce OsO 4 (Padham et al., 2007). There were more white plastoglobules in fib4 KD chloroplasts than wild-type chloroplasts, although the difference was not statistically significant (Fig. 1B). The total number of plastoglobules (electron transparent + electron opaque) was similar in fib4 KD and wild-type chloroplasts (Fig. 1C). Peripheral vesicles were similar in number in fib4 KD and wild-type chloroplasts (Fig. 1D). The peripheral vesicles were electron transparent, irregularly shaped, located adjacent to the chloroplast inner envelope membrane, and clearly distinguishable from the round, white plastoglobules (Fig. 1E). Black plastoglobules had varying patterns of electron opacity ranging from partially to fully electron opaque ( Fig. 1, A, F, and G). Black and white plastoglobules were often observed in the same chloroplast, sometimes in juxtaposition (Fig. 1H).

Photosynthetic CO 2 Fixation
Since fib4 KD apple tree plastoglobules exhibited altered ultrastructure, we decided to determine whether knockdown of FIB4 affected photosynthesis rates. Net photosynthetic CO 2 assimilation rates in wild-type and fib4 KD apple trees were compared under various levels of incident photosynthetically active radiation (PAR; Fig. 2). At midrange levels of PAR (300 and 600 mE m 22 s 21 ), no significant differences were detected between the net CO 2 fixation rates of wild-type and fib4 KD trees. Under high levels of PAR (1,000 mE m 22 s 21 ), however, wild-type trees had a significantly higher net CO 2 fixation rate than fib4 KD trees. Surprisingly, under low levels of PAR (100 mE m 22 s 21 ), the situation was reversed, with fib4 KD trees having a significantly higher net CO 2 fixation rate than wild-type trees. No significant differences in stomatal conductance were observed between wild-type and  fib4 KD trees; however, differences in internal [CO 2 ] consistent with differences in net CO 2 assimilation rates were observed in fib4 KD compared with wildtype trees (Supplemental Fig. S3).

Sensitivity to Increases in Light Intensity
The results of the photosynthesis measurements suggested that fib4 KD apple trees were sensitive to high light. Plants typically accumulate anthocyanins in response to high light stress as photoprotectants (for review, see Steyn et al., 2002). Under a light intensity of 90 mE m 22 s 21 in plant growth chambers with a 12-h photoperiod, the leaves of fib4 KD and wild-type apple trees were green and had similar levels of anthocyanins, chlorophyll a, chlorophyll b, and carotenoids ( Fig. 3A; Supplemental Fig. S4). However, following transfer to 600 mE m 22 s 21 light intensity conditions with a 12-h photoperiod for 6 d, fib4 KD tree leaves were visibly darker in color than leaves of wild-type trees (Supplemental Fig. S4) and accumulated higher amounts of anthocyanins than wild-type tree leaves, while levels of other pigments were similar (Fig. 3B).

Methyl Viologen Sensitivity
The sensitivity of fib4 KD apple trees to high light and increases in light intensity led us to suspect that they might be more sensitive to ROS generation in the chloroplast. Therefore, we tested the sensitivity of fib4 KD apple trees to methyl viologen (MV). MV generates ROS in chloroplasts by preferential electron acceptance by MV, rather than by ferredoxin, from the reaction center of PSI, producing MV radical, whose oxidation by oxygen produces the superoxide radical O 2 .- (Izawa, 1980;Ashton and Crafts, 1981;Taiz and Zeiger, 2006), thereby inducing oxidative damage. When floated on 0.5 mM MV solution for 24 h, leaf discs from fib4 KD apple trees developed obviously larger areas of brown pigmentation compared with leaf discs from wild-type and VC trees (Fig. 4A). Little or no brown pigmentation was observed on most of the wild-type and VC leaf discs after MV treatment; in contrast, all fib4 KD leaf discs developed extensive brown coloration. When observed, brown pigmentation in wild-type and VC leaf discs was generally restricted to limited areas near the disc margins. In addition, tissue damage as measured by electrolyte leakage was greater in fib4 KD leaf discs than wildtype or VC leaf discs at 24 h after the start of MV treatment (Fig. 4B). The greater sensitivity to MV was accompanied by higher levels of superoxide during the course of MV treatment (Fig. 4C), as detected by in situ staining of leaf discs with nitroblue tetrazolium (NBT) using the method described by Garmier et al. (2007). At 3 h after the start of MV treatment, leaf discs from fib4 KD trees exhibited measurably darker NBT staining than leaf discs from wild-type trees (Fig. 4C). This indicates that fib4 KD trees had higher levels of superoxide during MV treatment than wild-type trees. Apple showed a high, stable background signal in the superoxide assay; however, the signal increase in fib4 KD was clear. These results suggest that fib4 KD trees were more sensitive to oxidative stress.

Susceptibility to Bacterial Pathogens
Oxidative damage also plays a role in disease processes, such as fire blight (Venisse et al., 2001). Therefore, the susceptibility of fib4 KD apple trees to E. amylovora was tested. One of the major symptoms of Figure 1. (Continued.) and D, 80 chloroplasts from six leaves (two leaves from each of three plants) were observed for each genotype; data are means 6 SE; * P , 0.05 using Student's t test. B, Wild-type chloroplasts had more electron-opaque (black) plastoglobules than fib4 KD chloroplasts; the numbers of electron-transparent (white) plastoglobules were not significantly different between wild-type and fib4 KD chloroplasts. Partially and completely black plastoglobules were counted as electron opaque; plastoglobules scored as electron transparent had no electron-opaque sectors. C, Wild-type and fib4 KD chloroplasts had similar total numbers of plastoglobules (electron opaque + electron transparent). D, Wild-type and fib4 KD chloroplasts had similar numbers of peripheral vesicles. E, Transmission electron micrograph of peripheral vesicles (V). F, Electron-opaque plastoglobule in a wildtype chloroplast with less than 50% of its area appearing black. G, Electron-opaque plastoglobule in a wild-type chloroplast with more than 50% of its area appearing black. H, Adjacent electron-opaque and electron-transparent plastoglobules in a fib4 KD chloroplast. Figure 2. Effects of FIB4 knockdown on apple tree net photosynthetic CO 2 assimilation rates. Net CO 2 assimilation rates (mmol CO 2 m 22 s 21 ) in wild-type (WT) and fib4 KD apple tree leaves at various levels of PAR are shown. For each genotype, measurements were taken from three leaves from each of five plants growing in a greenhouse with natural photoperiod and light intensity. Data are means 6 SE; * P , 0.05 using Student's t test.
fire blight is a zone of darkened, shriveled, necrotic tissue that develops and spreads systemically from a point of initial infection. Necrotic region length as a percentage of total shoot length is a standard, quantitative measure of fire blight disease severity (Lee et al., 2010). Wild-type, VC, and fib4 KD apple trees growing in soil in a growth chamber were inoculated with 10 8 colony-forming units (cfu) mL 21 E. amylovora strain Ea581a at wounded shoot tips. Fire blight disease severity was measured once each week for 3 weeks following inoculation. At each time point, fire blight disease severity was significantly higher in fib4 KD apple trees than in either wild-type or VC trees (Fig. 5A). In addition, fib4 KD apple tree shoots supported significantly larger E. amylovora population sizes than wild-type shoots (Fig. 5B). These results indicated that fib4 KD apple trees were more susceptible to E. amylovora than control trees.
To determine whether plant immune responses were altered in fib4 KD apple trees during fire blight infection, we analyzed the expression of Pathogenesis-Related (PR) genes in E. amylovora-infected apple trees. PR gene expression is induced in apple trees during E. amylovora infection (Venisse et al., 2002). Our results indicated that expression of PR2 and PR8 was induced in control and fib4 KD plants; however, induction was significantly less in fib4 KD apple trees compared with wild-type trees (Supplemental Fig. S5).
To further study the role of FIB4 in disease resistance, effects of mutations in the orthologous Arabidopsis FIB4 gene (At3g23400) on bacterial disease resistance were determined. For these experiments, two transgenic Arabidopsis lines homozygous for T-DNA mutations in FIB4 (fib4-1 and fib4-2) were obtained and characterized (Supplemental Fig. S6). Pseudomonas syringae pv tomato (P. s. t.) causes bacterial speck disease in Arabidopsis (Whalen et al., 1991). Symptoms observed on the leaves of susceptible Arabidopsis plants include tissue necrosis, watersoaked lesion development, and chlorosis. Wild-type ecotype Columbia-0 (Col-0) and fib4-1 mutant plants were challenged with the virulent P. s. t. strain DC3000 by leaf infiltration, and symptom development and bacterial growth were monitored. Bacterial speck disease symptoms were visibly more severe on fib4-1 than Col-0 at 6 d post inoculation (dpi) with 10 4 cfu mL 21 P. s. t. (Fig. 5C). Significantly greater electrolyte leakage was recorded for fib4-1 than Col-0 at 6 dpi with 10 4 cfu mL 21 P. s. t. (Fig. 5D), indicating more extensive tissue damage in the mutant. In addition, the P. s. t. population size in fib4-1 plant leaves was double that in Col-0 leaves at 4 dpi with 10 4 cfu mL 21 bacterium (Fig. 5E). These results show that the Arabidopsis fib4-1 mutant was more susceptible to bacterial speck disease than wild-type control plants, although the change in bacterial growth was relatively modest compared with some other Arabidopsis mutants that support increased P. s. t. growth, such as sid2 (Block et al., 2005), eds1, and pad4 (Xing and Chen, 2006).

Ozone Sensitivity
The sensitivity of fib4 KD trees to MV, high light intensity, and bacterial pathogens prompted us to test whether fib4 KD trees were sensitive to ozone (O 3 ), another oxidative stress. Exposure to O 3 triggers tissue necrosis and cell death in sensitive plants (Runeckles and Chevonne, 1992). Wild-type and fib4 KD apple trees were exposed to 350 nL L 21 O 3 for 6 h. At 6 h after the start of the O 3 treatment, none of the apple trees had Figure 3. Effects of FIB4 knockdown on apple tree leaf major pigment levels. A, Major pigment levels in leaves of wild-type (WT) and fib4 KD plants grown in a growth chamber with 90 mE m 22 s 21 light intensity and a 12-h photoperiod. Data are from one representative experiment; data are means 6 SE; n = 6 per genotype/experiment. No statistically significant differences at P , 0.05 between wild-type and fib4 KD plants were observed for any pigment under these conditions using Student's t test. B, Major pigment levels in leaves of wild-type and fib4 KD plants at 6 d after transfer from a growth chamber with 90 mE m 22 s 21 light intensity and a 12-h photoperiod to a growth room having 600 mE m 22 s 21 light intensity and a 12-h photoperiod. Data are means 6 SE; n = 6 per genotype. FW, Fresh weight. necrotic lesions. However, at 24 h after the start of O 3 treatment, fib4 KD apple trees developed extensive necrosis, especially in the older leaves, while wildtype and VC trees displayed little or no necrosis (Fig.  6A). Tissue damage as measured by electrolyte leakage was significantly greater in fib4 KD apple trees than in wild-type and VC apple trees at 24 h after the start of the O 3 treatment (Fig. 6B). These results indicate that fib4 KD apple trees were much more sensitive to O 3 than control wild-type and VC trees.
Arabidopsis fib4 mutants were also more sensitive to O 3 . Exposure of homozygous Arabidopsis fib4-1 mutants and Col-0 control plants to 500 nL L 21 O 3 for 6 h or 700 nL L 21 O 3 for 3 h resulted in higher numbers of leaves with visible lesions per plant in fib4-1 than in Col-0 at 24 h after the start of O 3 exposure (Fig. 6, C and  D). Tissue damage as measured by electrolyte leakage was greater in fib4-1 and fib4-2 mutant plants than in Col-0 plants at 6 h after the start of a 3-h, 500 nL L 21 O 3 treatment ( Fig. 6E; Supplemental Fig. S7). These re-sults indicate that Arabidopsis fib4-1 and fib4-2 mutant plants were more sensitive to O 3 than Col-0, which was consistent with the increased O 3 sensitivity of fib4 KD apple trees.

Plastoglobule Ultrastructure during O 3 Stress
In order to determine whether O 3 stress caused any changes in plastoglobule ultrastructure, wild-type and fib4 KD apple trees were exposed to 350 nL L 21 O 3 for 6 h, leaf tissue was collected and fixed at 2 d after the beginning of O 3 exposure, and plastoglobules in palisade mesophyll cells were examined by TEM. In wildtype plants, the proportion of electron-transparent plastoglobules increased from 4% of total plastoglobules before O 3 treatment to 43% of total plastoglobules after O 3 treatment (Fig. 7, A and B). There were fewer electron-opaque plastoglobules and more total plastoglobules in wild-type plants after O 3 treatment, although the differences were not statistically signifi-  (Garmier et al., 2007). NBT staining intensity was measured using ImageJ software (Abramoff et al., 2004). The experiment was performed using leaf discs from five trees per genotype. Statistical significance was determined using Student's t test. cant (Fig. 7B). In fib4 KD plants, electron-opaque plastoglobules were virtually absent both before and after O 3 treatment, as expected; however, the total number of plastoglobules in fib4 KD increased by 56% after O 3 treatment due to an increase in the number of electron-transparent plastoglobules (Fig. 7, A and C).

DISCUSSION
This study indicates that FIB4 is required for plant resistance to several abiotic stresses and for resistance to bacterial pathogens. This finding is consistent with the growing body of evidence linking fibrillins with stress tolerance and disease resistance (Pruvot et al., 1996;Gillet et al., 1998;Rey et al., 2000;Rorat et al., 2001;Cooper et al., 2003;Leitner-Dagan et al., 2006;Yang et al., 2006;Youssef et al., 2010). It is interesting that FIB4 was found to be required for resistance to P. s. t., as FIB4 is one of a handful of proteins that is phosphorylated early on during defense responses against this bacterium, suggesting a role for FIB4 in basal defenses (Jones et al., 2006). The phenotypic similarities between Arabidopsis fib4 mutants and fib4 KD apple trees indicate that the function of FIB4 is conserved between these two very different species. This implies that FIB4 may play a basic, conserved role in plant physiology, perhaps as a contributor to plant stress tolerance and/or photosynthetic acclimatization.
This study also indicates that FIB4 is involved in plastoglobule development. While FIB4 does not appear to be required for the production of plastoglobules, the dramatic decrease in plastoglobule osmiophilicity in fib4 KD apple tree leaf chloroplasts compared with the wild type under benign conditions suggests that FIB4 is required for the development of plastoglobule osmiophilic content. The reactivity of Figure 5. Knockdown or mutation of FIB4 causes increased susceptibility to bacterial pathogens in apple and Arabidopsis. A and B represent experiments done in apple; C, D, and E represent experiments done in Arabidopsis. All plants were maintained under 90 mE m 22 s 21 light intensity, with a 12-and 10-h photoperiod for apple and Arabidopsis, respectively, for the duration of the experiments. A, Fire blight disease severity (necrotic zone size) in wild-type (WT), VC, and fib4 KD trees at 7, 14, and 21 dpi with 10 8 cfu mL 21 E. amylovora. Data are from one representative experiment; the experiment was repeated four times with similar results. Data are means 6 SE; n = 8 per genotype/experiment; * P , 0.05 using Student's t test. B, E. amylovora population levels in wild-type and fib4 KD tree stems at 7 dpi. Data are means 6 SE; n = 4 or more per genotype; * P , 0.05 using Student's t test. C, Symptom development on Col-0 and fib4-1 plant leaves at 6 dpi with 10 4 cfu mL 21 P. s. t. D, Ion leakage from Col-0 and fib4-1 leaves at 6 dpi with 10 4 cfu mL 21 P. s. t. Data are from one representative experiment; the experiment was repeated three times with similar results. Data are means 6 SE; n = 5 per genotype/experiment; * P , 0.05 using Student's t test. E, P. s. t. population levels in Col-0 and fib4-1 mutant plant leaves at 4 dpi with 10 4 cfu mL 21 bacterium. Data are from one representative experiment; the experiment was repeated three times with similar results. Data are means 6 SE; n = 5 per genotype/ experiment; * P , 0.05 using Student's t test. osmium oxides with certain biological materials was noted upon the discovery of osmium (Tennant, 1804). Some major plastoglobule constituents such as plastoquinone, carotenoids (Bahr, 1954), and triacylglycerides are expected to contribute to plastoglobule osmiophilicity because their ethylenic double bonds reduce OsO 4 , probably producing stable diester adducts (Wigglesworth, 1957) that scatter electrons due to the high mass density and atomic number of osmium (Valentine, 1958). In addition, the reducing power of tocopherols and plastoquinone may contribute to plastoglobule osmiophilicity (Dahlin and Ryberg, 1986). Under mild conditions, FIB4 appears to play a role in the accumulation of osmiophilic material within plastoglobules. However, FIB4 cannot be the sole factor required for plastoglobule osmiophilicity, since osmiophilic plastoglobules were observed in fib4 KD trees, albeit much fewer than in wild-type apples.
Our finding that plastoglobule osmiophilicity decreased in wild-type apple chloroplasts following acute O 3 stress was consistent with the reduced plastoglobule osmiophilicity observed in earlier studies of chronic O 3 exposure in radish (Raphanus sativus; Miyake et al., 1989) and Norway spruce (Picea abies; Kivimäenpää et al., 2003). Decreased plastoglobule osmiophilicity could reflect oxidation (Miyake et al., 1989) or egress of plastoglobule osmiophilic content during O 3 stress. This suggests that plastoglobules may be dynamic reservoirs of antioxidants that dispense their contents as needed during O 3 stress; fib4 KD plastoglobules, which contain very little osmiophilic material to begin with, may be unable to perform this function. This could account for the increased O 3 sensitivity and oxidative stress sensitivity of fib4 KD trees compared with wild-type trees. In addition, the increase in total number of plastoglobules in fib4 KD during O 3 stress may represent a physiological compensation for a dearth of osmiophilic plastoglobules.
The increased sensitivity of fib4 KD plants to MV implies that they are more sensitive to oxidative stress, particularly in the chloroplast. The higher photosynthesis rates of fib4 KD trees compared with wild-type trees under low-light conditions, which were used for the MV treatments, would likely have exacerbated oxidative stress due to MV treatment in fib4 KD trees. The higher level of superoxide in MV-treated fib4 KD apple leaves than in wild-type apple leaves is consistent Figure 7. Plastoglobule ultrastructure changes in wild-type and fib4 KD following O 3 exposure. Wild-type (WT) and fib4 KD apple trees were exposed to 350 nL L 21 O 3 for 6 h under 90 mE m 22 s 21 light intensity and then returned to their normal growth conditions (90 mE m 22 s 21 , 10-h photoperiod). Leaf tissues were collected at 2 d after the beginning of the O 3 exposure and examined by TEM. Data shown are from one experiment; the experiment was performed twice with similar results each time. A, Transmission electron micrographs of representative plastoglobules (arrows) in control and O 3 -treated wild-type and fib4 KD leaf palisade mesophyll cell chloroplasts. The scale is the same in all four micrographs. B, Numbers of electron-transparent plastoglobules (white), electron-opaque plastoglobules (black), and total plastoglobules (black + white) per mm 2 in wild-type chloroplasts before and after O 3 exposure. Plastoglobules were counted in 14 chloroplasts from a control plant and 11 chloroplasts from an O 3 -exposed plant. Data are means 6 SE; * P , 0.05 using Student's t test. C, Numbers of black and white plastoglobules and total plastoglobules per mm 2 in fib4 KD chloroplasts before and after O 3 exposure. Plastoglobules were counted in 15 chloroplasts from a control plant and 15 chloroplasts from an O 3exposed plant. Data are means 6 SE; * P , 0.05 using Student's t test.
with this scenario. Increases or decreases in chloroplast ROS sensitivity are known to affect MV sensitivity. For example, when chloroplastic ROS-scavenging capacity is inhibited by decreasing the expression of thylakoid ascorbate peroxidase (APX), MV sensitivity increases (Tarantino et al., 2005). Furthermore, increasing chloroplastic ROS-scavenging capacity by overexpressing chloroplastic APX (Murgia et al., 2004) or overexpressing cytosolic APX in the chloroplast (Badawi et al., 2004) reduces sensitivity to MV.
Higher anthocyanin accumulation in the leaves of fib4 KD apple trees than in wild-type apple trees upon shifting to higher light intensity further supports the notion that fib4 KD plants are more sensitive to oxidative stress in the chloroplast. Anthocyanins protect photosynthetic systems from high light by sunscreen and antioxidant activities (Smillie and Hetherington, 1999;Hughes et al., 2005;Kytridis and Manetas, 2006). Higher anthocyanin accumulation in the leaves of fib4 KD apple trees upon shifting to higher light intensity might partially compensate for a lack of FIB4-mediated control of oxidative stress in the chloroplast. In addition, increased photoinhibition in fib4 KD apple leaves compared with the wild type at high light intensity suggests a reduced ability to control photooxidative damage of the photosynthetic system in fib4 KD trees. The increased O 3 sensitivity of fib4 KD apple and fib4 Arabidopsis plants is also consistent with FIB4 contributing to chloroplast oxidative damage control, since chloroplastic ROS production is an early response of plants to O 3 exposure (Joo et al., 2005).
Similarly, the increased susceptibility of Arabidopsis fib4-1 mutant plants to bacterial speck disease is consistent with increased sensitivity to oxidative damage. P. s. t. induces light-dependent ROS accumulation in susceptible host tissues via the phytotoxic virulence factor coronatine (Ishiga et al., 2009). Furthermore, the chloroplast electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, which suppresses chloroplast ROS production (Joo et al., 2005), blocks bacterial speck disease development in tomato cotyledons (Ishiga et al., 2009). This suggests that ROS production in chloroplasts is required for bacterial speck disease development (Ishiga et al., 2009). Thus, FIB4 could contribute to bacterial speck disease resistance by mediating oxidative damage control in the chloroplast.
The increased susceptibility of fib4 KD apple trees to E. amylovora is consistent with an increased sensitivity to oxidative damage, although there is currently no clear link between the chloroplast and fire blight. E. amylovora induces superoxide production in host plants during infection, resulting in membrane damage and necrosis (Venisse et al., 2001). The production of superoxide appears to play a role in fire blight disease development, possibly by inducing cell death processes in the host. In addition, PR gene expression was partially suppressed in fib4 KD apple trees during E. amylovora infection. PR gene expression is normally induced in apple trees during E. amylovora infection (Venisse et al., 2002), and some of these genes could play a role in reducing apple susceptibility to E. amylovora (Maxson-Stein et al., 2002;Malnoy et al., 2007). These results indicate that fib4 KD apple trees had weakened resistance to E. amylovora. In light of these findings, it is interesting that the E. amylovorasecreted virulence protein HrpN (Wei et al., 1992) can physically associate with FIB4 (Song et al., 2002). Although this interaction has not been confirmed in vivo, it might make sense if FIB4 were a target of bacterial effector proteins, since reduction of FIB4 expression triggered disease susceptibility in this study.
The presence of FIB4 in the plastoglobule, the PSII light-harvesting complex, and the thylakoid membrane system, coupled with the presence of a conserved lipocalin signature in FIB4 (Jones et al., 2006), presents a possible mechanism for FIB4 function in plastoglobule development and oxidative stress. Based on its structural similarity to lipocalins (for review, see Flower et al., 2000), FIB4 may be hypothesized to interact with one or more plastoglobule lipid components, helping to transport lipophilic antioxidants in and out of the plastoglobule. In addition, FIB4 could be involved in the delivery of lipophilic antioxidants to particular locations within the photosynthetic membrane system, such as the PSII light-harvesting complex. When the amount of FIB4 is greatly reduced, such as in the fib4 KD apple trees, then lipophilic antioxidants may not be properly loaded into plastoglobules under benign conditions and may not be dispensed from plastoglobules to locations where they are needed during stress. This hypothesis would explain the increased sensitivity to oxidative stress and alterations in plastoglobule osmiophilicity observed in the fib4 KD apple trees.

Plant Material
Arabidopsis (Arabidopsis thaliana) plants were grown as described previously (Jambunathan et al., 2001). Arabidopsis T-DNA insertion mutants were obtained from the Arabidopsis Biological Resource Center, a publicly accessible collection of T-DNA insertion lines (http://www.biosci.ohio-state.edu/ pcmb/Facilities/abrc/abrchome.htm; Alonso et al., 2003). Transgenic RNAi and control pGH00.0131-EGFP expressing apple (Malus 3 domestica) plants were generated as described previously (Maximova et al., 1998). For details of vector construction and transformation to generate fib4 KD apple plants, see Supplemental Figure S1 and its legend. In vitro rooted apple plants were transferred to potting mix (Redi-Earth) and acclimated in a growth chamber under the same environmental conditions as described for Arabidopsis (Jambunathan et al., 2001).

RNA Extraction, DNA Extraction, and PCR
Total RNA from apple leaves was isolated using an established protocol (Jensen et al., 2010). Total RNA from Arabidopsis leaves was isolated as described previously (Jambunathan et al., 2001). DNA was eliminated from RNA samples using the DNA-Free kit (Ambion). Conventional reverse transcription-PCR was performed as described previously (Liu et al., 2005; for primers, see Supplemental Table S1). Quantitative real-time PCR analyses were performed at the Pennsylvania State University Genomics Core Facility as described previously (Jensen et al., 2010; for primers and probes, see Supplemental Table S2). For PR gene expression analyses, apple inoculations with Erwinia amylovora and tissue collection were done as described by Norelli et al. (2009) using gene-specific primers for each PR gene (for primers, see Supplemental Table S2). Genomic DNA was isolated from young leaves using standard methods (Kasajima et al., 2004). PCR was performed using the Advantage 2 PCR enzyme system kit according to the manufacturer's instructions (Clontech). The LBb1.3 primer sequence was obtained from http:// signal.salk.edu/tdnaprimers.html.

TEM
For the TEM experiments presented in Figure 1, leaf discs were collected from fully expanded leaves of 4-to 5-month-old apple plants acclimated in the growth chamber with 90 mE m 22 s 21 light intensity and a 12-h photoperiod. Tissues were fixed with glutaraldehyde in HEPES buffer, postfixed in OsO 4 in the same buffer, and embedded in Spurr's resin, according to the method of Owen and Makaroff (1995), essentially as described by Li et al. (2004). Ultrathin sections (80 nm thick) were cut using a Reichart-Jung Ultracut E Microtome and stained with uranyl acetate and lead citrate. Observations of chloroplasts were made with a JEOL 1200 EX II transmission electron microscope fitted with a high-resolution Tietz F224 digital camera. Chloroplast areas were measured using ImageJ software (http://rsbweb.nih.gov/ij/). Plastoglobules were counted independently by two people, and one count was done as a blind experiment. For the O 3 TEM experiments presented in Figure 7, 1-to 2-month-old apple trees growing in a growth room with 90 mE m 22 s 21 light intensity and a 10-h photoperiod were exposed to 350 nL L 21 O 3 for 6 h. Leaf tissues were collected at 2 d after the beginning of the O 3 exposure and examined by TEM, and plastoglobules were counted.

Photosynthesis Measurements
Photosynthesis measurements were carried out on potted fib4 KD and wild-type trees maintained at 22°C 6 3°C in a greenhouse. The second, third, and fourth fully expanded leaves on each of five replicate trees were used for measurements, and the readings were repeated three times for each of the experiments. Measurements were taken on the center of each leaf. For both light intensity and intercellular [CO 2 ] experiments, measurements were taken on consecutive days between 10:00 AM and 2:00 PM on each day. Gas-exchange measurements were taken with a CIRAS-I infrared gas analyzer equipped with a 2.5-cm 2 Parkinson (PLC 6U) leaf cuvette with light-emitting diode lights (PP Systems). Light at the indicated intensity was applied to the leaves, and net CO 2 assimilation, stomatal conductance, and intercellular [CO 2 ] were measured.

Pigment Analysis
Anthocyanins, carotenoids, chlorophyll a, and chlorophyll b were extracted from leaves and measured as described by Sims and Gamon (2002).

Light, O 3 , and Herbicide Treatments
O 3 treatments were performed in a tissue culture chamber (model CU-32L; Percival Scientific) attached to an O 3 generator (model 2000; Jelight Company) and an O 3 monitor (model 450; Advanced Pollution Instrumentation). Highlight treatment consisted of exposure to 600 mE m 22 s 21 for 6 d at 21°C with a 12-h photoperiod and 35% to 45% relative humidity. For herbicide treatments, apple tree leaf discs were cut with a 1-cm-diameter cork borer and floated on 20 mL of water or 0.5 mM MV (Sigma) in a petri dish for 24 h. Fifteen leaf discs representing two individual plants (seven or eight discs per plant) were used per sample, and six biological replicates were done for each plant genotype for each treatment.

Disease Assays
Pseudomonas syringae infection and population analysis in Arabidopsis was done using a published protocol (Jambunathan et al., 2001). Apple infection with E. amylovora and disease severity measurement was done as described previously (Jensen et al., 2003). Bacterial populations in apple tissue were determined by tissue extraction and serial dilution plating.

Electrolyte Leakage Assays
For the MV experiments, the conductivity of the MV treatment and control solutions (in mS cm 21 ) was measured at 24 h after the beginning of the experiment using a Corning 316 conductivity meter and divided by the leaf area used in the sample (in m 2 ). Relative ion leakage (Joo et al., 2005) was used for the bacterial speck disease and O 3 experiments. Apple or Arabidopsis leaves were collected, rinsed with deionized water, and immersed in 25 mL of deionized water for 4 h with shaking at 100 rpm, and the conductivity (mS cm 21 ) of the resulting solution was measured. The samples were then autoclaved, and conductivity was measured again. Relative ion leakage was calculated by dividing the conductivity of the electrolyte leakage solution before autoclaving by the conductivity of the electrolyte solution after autoclaving and multiplying by 100.

Detection of Superoxide
In situ O 2 .was detected using a NBT staining method essentially as described by Garmier et al. (2007). In brief, MV-treated leaf discs were vacuum infiltrated with a solution of 0.5 mg mL 21 NBT (Sigma) in 10 mM potassium phosphate buffer (pH 7.8) and kept in dark for 1 h, and then chlorophyll was cleared by immersion in ethanol at 70°C. Leaf discs were photographed using a FinePix S9000 digital camera (FujiFilm), and NBT staining intensity was measured using ImageJ software (Abramoff et al., 2004). Each sample consisted of five leaf discs, each disc taken from a different tree.

Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Construction of the apple FIB4 RNAi binary vector used for Agrobacterium tumefaciens-mediated genetic transformation of apple.
Supplemental Figure S2. Confirmation of reduced expression of the FIB4 gene in fib4 KD apple trees, and phenotype of wild-type and transgenic trees.
Supplemental Figure S3. Internal [CO 2 ] and stomatal conductance of fib4 KD and wild-type apple trees.
Supplemental Figure S4. Effects of transfer from 90 mE m 22 s 21 PAR to 600 mE m 22 s 21 PAR for 6 d on wild-type and fib4 KD plants.
Supplemental Figure S5. Expression of PR genes was induced in wildtype (WT) and fib4 KD apple tree during E. amylovora infection.
Supplemental Figure S6. Confirmation of T-DNA insertion in the Arabidopsis FIB4 gene.
Supplemental Table S2. Real-time PCR primer and probe sequences.