University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln

Biochemistry -- Faculty Publications Biochemistry, Department of

2013 Cytochrome b5 Reductase Encoded by CBR1 Is Essential for a Functional Male Gametophyte in Arabidopsis Laura L. Wayne Washington State University

James G. Wallis Washington State University

Rajesh Kumar Washington State University

Jonathan E. Markham University of Nebraska-Lincoln, [email protected]

John Browse Washington State University, [email protected]

Follow this and additional works at: http://digitalcommons.unl.edu/biochemfacpub

Wayne, Laura L.; Wallis, James G.; Kumar, Rajesh; Markham, Jonathan E.; and Browse, John, "Cytochrome b5 Reductase Encoded by CBR1 Is Essential for a Functional Male Gametophyte in Arabidopsis" (2013). Biochemistry -- Faculty Publications. 157. http://digitalcommons.unl.edu/biochemfacpub/157

This Article is brought to you for free and open access by the Biochemistry, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Biochemistry -- Faculty Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. The Plant Cell, Vol. 25: 3052–3066, August 2013, www.plantcell.org ã 2013 American Society of Plant Biologists. All rights reserved.

Cytochrome b5 Reductase Encoded by CBR1 Is Essential for a Functional Male Gametophyte in ArabidopsisC W

Laura L. Wayne,a,1 James G. Wallis,a Rajesh Kumar,a,2 Jonathan E. Markham,b and John Browsea,3 a Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340 b Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588

In all eukaryotes, NADH:cytochrome b5 reductase provides electrons, via cytochrome b5, for a range of biochemical reactions in cellular metabolism, including for fatty acid desaturation in the endoplasmic reticulum. Studies in mammals, yeast, and in vitro plant systems have shown that cytochrome b5 can, at least in some circumstances, also accept electrons from NADPH:cytochrome P450 reductase, potentially allowing for redundancy in reductase function. Here, we report characterization of three T-DNA insertional mutants of the encoding cytochrome b5 reductase in Arabidopsis thaliana, CBR1. The progeny of plants heterozygous for the cbr1-2 allele segregated 6% homozygous mutants, while cbr1-3 and cbr1-4 heterozygotes segregated 1:1 heterozygous:wild type, indicating a gametophyte defect. Homozygous cbr1-2 seeds were deformed and required Suc for successful germination and seedling establishment. Vegetative growth of cbr1-2 plants was relatively normal, and they produced abundant flowers, but very few seeds. The pollen produced in cbr1-2 anthers was viable, but when germinated on cbr1-2 or wild-type stigmas, most of the resulting pollen tubes did not extend into the transmitting tract, resulting in a very low frequency of fertilization. These results indicate that cytochrome b5 reductase is not essential during vegetative growth but is required for correct pollen function and seed maturation.

INTRODUCTION 20- and 22-carbon polyunsaturated fatty acids required for hu- man health (Wallis et al., 2002). In the ER of plant cells, FATTY The transfer of electrons is fundamental to metabolism within ACID DESATURASE2 (FAD2) converts oleic acid (18:1) to 18:2 the cell. Distinct from the chloroplast and mitochondrial (Miquel and Browse, 1992; Okuley et al., 1994), followed by pathways, there are two prominent electron transfer systems in conversion of 18:2 to 18:3 by the FAD3 linoleate desaturase the endoplasmic reticulum (ER) membrane. One operates through (Browse et al., 1993). NADPH:cytochrome P450 reductase, transferring electrons to the Mammalian cytochrome b5 reductase (EC 1.6.2.2) is an ER many cytochrome P450 monooxygenases, and the other oper- flavoprotein that transfers electrons from NADH to the FAD ates through NADH:cytochrome b5 reductase, transferring elec- prosthetic group of the reductase and subsequently to the heme trons to cytochrome b5 proteins. In plants, a primary role for the of cytochrome b5 (Percy and Lappin, 2008). The crystal struc- cytochrome b5 electron transfer system is to supply reductant for ture shows two cytoplasmic domains, a FAD binding domain the synthesis of the polyunsaturated fatty acids linoleic acid (18:2) and a NADH binding domain, joined by a linker region and a and -linolenic acid (18:3) (Slack et al., 1976; Kearns, et al., 1991; a single transmembrane region at the N terminus (Bando et al., Shanklin and Cahoon, 1998), which are integral to the function of 2004; Bewley et al., 2001). These structural components are cellular membranes (McConn and Browse, 1998). Synthesis of strongly conserved in the family of NADH:cytochrome b5 re- polyunsaturated fatty acids occurs in all plant tissues, and in ductases, including CBR1 of Arabidopsis. The links that follow developing seeds of Arabidopsis thaliana, they make up half of CBR1 in electron transport are the five Arabidopsis cytochrome triacylglycerol, the storage lipid that is the principal component of b5 proteins (CB5-A to CB5-E) and one CB5-like protein (Nagano seed oil. Human consumption of triacylglycerol extracted from et al., 2009); four of these are predicted to localize to the ER oilseed crops provides 18:2 and 18:3 fatty acids that are both membrane (Hwang et al., 2004; Nagano et al., 2009). Although essential dietary components and precursors for the synthesis of the specific functions of individual CB5 proteins are unknown, there is likely to be some degree of redundancy. Several en- 1 Current address: Dow AgroSciences, 9330 Zionsville Road, Indian- zymes also contain cytochrome b5 domains (Sperling, et al., apolis, IN 46268. 1998). A second Arabidopsis cytochrome b5 reductase, CBR2, 2 Current address: National Research Center on DNA Fingerprinting, is localized to the inner mitochondrial membrane, and there is no National Bureau of Plant Genetic Resources, New Delhi, India 110012 evidence that it is part of the ER electron transport system 3 Address correspondence to [email protected]. The author responsible for distribution of materials integral to the findings (Heazlewood et al., 2004). presented in this article in accordance with the policy described in the Whereas fatty acid desaturation is a major function of electron Instructions for Authors (www.plantcell.org) is: John Browse (jab@wsu. transfer through cytochrome b5, additional of primary edu). and secondary metabolism also rely on cytochrome b5 to provide C Some figures in this article are displayed in color online but in black and electrons. These include enzymes contributing to the synthesis white in the print edition. W Online version contains Web-only data. and modification of cutin, suberin, cuticular waxes, sterols, and www.plantcell.org/cgi/doi/10.1105/tpc.113.113324 sphingolipids (Boutte and Grebe, 2009; Li-Beisson et al., 2013; CBR1 and Male Gametophyte Function 3053

Markham, et al., 2013). In sphingolipid metabolism, there are five type Arabidopsis (Kumar et al., 2006). To characterize the effects cytochrome b5–dependent hydroxylation and desaturation re- of complete loss of function in CBR1, we obtained several actions that contribute to the synthesis of the predominant gly- T-DNA insertional alleles of cbr1 from the ABRC and Gabi-Kat cosyl inositolphosphoceramides and glucosylceramides that are (Rosso et al., 2003). After preliminary analysis (see Methods for important components of the plasma membranes in plant cells details), three alleles were selected for further characterization. (Markham, et al., 2013). PCR analysis of Sail_644_A11, renamed cbr1-2, revealed a tan- CBR1-mediated electron transfer through cytochrome b5 to dem insertion located in the middle of three, with two left fatty acid desaturases may not be the only electron sources for borders facing genomic DNA; 28 nucleotides of genomic DNA some of these enzymes. Reports indicate that NADPH:cyto- are unaccounted for between the two left borders (Figure 1A). chrome P450 reductase, encoded by ARABIDOPSIS THALIANA The left border of this insertion that is oriented toward the 39 end REDUCTASE1 (ATR1) and ATR2 in Arabidopsis, can transfer of the gene is described correctly in the SIGnAL T-DNA Express electrons from NADPH to cytochrome b5; these electrons would database (http://signal.salk.edu/cgi-bin/tdnaexpress) (Alonso be available to enzymes of the cytochrome b5 pathway (Fukuchi- et al., 2003). Our sequence analysis shows that the WiscDsLox Mizutani et al., 1999). In mammals, there is often functional 377-380021 insertion (Woody et al., 2007), renamed cbr1-3,is overlap between CBR1 and NADPH:cytochrome P450 re- located in the second to last nucleotide at the end of exon six ductase, for example, when NADPH-dependent reductase (Figure 1A), not in the following intron as described in the proteins supply electrons to cytochrome b5 in microsomal SIGnAL database. Finally, the Gabi-Kat 371A08 insertion, re- preparations (Enoch and Strittmatter, 1979; Dürr et al., 2007). named cbr1-4, is a tandem insertion with two left borders facing Genetic experiments demonstrate nearly complete redundancy genomic DNA, one left border in exon two and the other in intron of the two electron transfer systems in yeast (Saccharomyces one; 188 nucleotides of genomic DNA are unaccounted for cerevisiae). Yeast with null mutations in either Cbr1 or Ncp1 between the two left borders (Figure 1A). (YHR042W, the yeast ATR homolog) are viable, with somewhat slower growth, whereas the double null mutations are lethal cbr1 Mutants Have Distorted Segregation (Tiedje et al., 2007). Strong in vivo evidence for redundancy in electron transfer systems comes from the effects of a point These three T-DNA lines, obtained from the stock centers as mutation in Arabidopsis CBR1 that severely reduces its activity segregating T2 seeds, were germinated on agar medium and but only produces a decrease in 18:3 in seed fatty acid; aside transferred to soil; cbr1-2 and cbr1-3 plants were treated with from that effect, the mutant plants are indistinguishable from the Basta to select for the T-DNA insertions. Plants of all three lines wild type in growth, development, and seed production (Kumar were genotyped by PCR in an attempt to identify homozygous et al., 2006). This implies that, apart from a fairly subtle change in individuals. In the T2 generation, we found that cbr1-2, cbr1-3, oil formation, CBR1 is not critical to plant health. Since in many and cbr1-4 did not segregate homozygous plants (Table 1), cases electrons can be supplied to cytochrome b5 proteins and so heterozygous plants from each line were allowed to self- P450 enzymes either from NADH, via cytochrome b5 reductase, pollinate and their progeny were genotyped. For the cbr1-2 or from NADPH, via P450 reductase, it seems likely that the mutant, of 106 plants screened, only six homozygous plants P450 reductase could support normal Arabidopsis growth in the were identified in the T3 generation, representing 6% of the total absence of CBR1, similar to the phenotype seen in yeast. (Table 1, Figure 1B), significantly lower than the 25% predicted In contrast with the hypothesis that CBR1 is dispensable, we for Mendelian segregation. In this segregating T3 population of report here that three CBR1 T-DNA insertional knockout lines cbr1-2, 49% of the plants were heterozygous for the cbr1-2 suffered distorted segregation, with greatly reduced penetrance insert and 45% were wild type (Table 1). When the T3 progeny of through the male gametophyte. It was possible to identify plants heterozygous cbr1-3 or cbr1-4 plants were analyzed, only 46% homozygous for only one of the three mutations, cbr1-2, and of the cbr1-3 and 49% of the cbr1-4 carried the T-DNA insertion; for that line only 6% of the progeny were homozygous when no line homozygous for either of these two insertions could a heterozygote was allowed to self-pollinate. Homozygous cbr1-2 be obtained (Table 1). The cbr1-3 and cbr1-4 mutant lines plants produced very few seeds, and these exhibited reduced 18:3, continued to segregate in this fashion in the T4 generation; decreased total oil content, and delayed germination. In fertilization no homozygous plants from either allele were identified experiments, pollen of cbr1-2 plants germinated on wild-type and (Figure 1B, Table 1), even when more than 500 progeny from cbr1-2 stigmas, but the majority of pollen tubes stopped growing cbr1-3 heterozygotes were screened. Because the initial prior to reaching the ovules, leading to severely reduced fertilization segregating cbr1-4 T2 seedlings exhibited 98% resistance to and seed set. Our work reveals that, in Arabidopsis, CBR1 is re- sulfadiazine (the selectable marker of the T-DNA), indicating quired for pollen tube growth and normal fertilization. multiple T-DNA insertions, a backcross to wild-type Arabi- dopsis was necessary (described in Methods). From the RESULTS backcross, a heterozygous cbr1-4 plant was isolated in which 50% of the self-pollinated progeny were resistant to Identification of Three T-DNA Insertion Alleles in CBR1 sulfadiazine, but again no homozygous plant was detected. The distorted segregation observed in all of these T-DNA The ethyl methanesulfonate–generated mutant cbr1-1 has de- lines, with approximately a 1:1 segregation of heterozygous creased levels of 18:3 due to instability of the mutated CBR1 to the wild type, suggests that absence of CBR1 results in protein and reduced enzymatic activity but is otherwise like wild- a gametophytic defect. 3054 The Plant Cell

Figure 1. Isolation of a Homozygous cbr1-2 Mutant.

(A) Schematic of three T-DNA insertional mutations (triangles) cbr1-2, cbr1-3,andcbr1-4 located in the (black boxes) of CBR1, and the cbr1-1 point mutation (vertical line) previously characterized (Kumar et al., 2006). The locations of primers P1 to P7 are shown. (B) Results from PCR analysis of cbr1-2 show the tandem insertions (overlapping triangles in [A]) by amplification with each gene-specific primer, P1 or P2, and the left border primer, LB3. The P1+P2 primer combination amplified a product from wild-type DNA (WT) but not from DNA from a cbr1-2 homozygous plant. Comparable PCR analysis of cbr1-3 (primers P3, P4, and WiscLB) and cbr1-4 plants (primers P1, P2, 59PRM, and GKLB) only identified heterozygous individuals, containing both wild-type and T-DNA insertion alleles. (Primer 59PRM used for the amplification of cbr1-4 is located 828 bp upstream of the start codon and is not shown in [A].) (C) RT-PCR analysis of CBR1 transcript levels in cbr1-2 and the wild type shows there is no full-length transcript (primers P5+P6); however, there is a stable transcript downstream of the cbr1-2 insertion (primers P7+P6). ACT8 was amplified as a control for comparable RNA concentrations. In the two left lanes, no template DNA was added to the assay as a negative control.

The full siliques observed on heterozygous cbr1 mutant plants 1:1 (heterozygous:wild type). When each of the heterozygous suggested that the female gametophyte was functional, but to mutant lines was fertilized with the pollen of wild-type plants define which gametophyte was affected in these mutants, we (CBR1/cbr1 3 wild type) this expectation was met: cbr1 female reciprocally backcrossed each of the three cbr1 heterozygous gametophytes were functional. However, for the reciprocal mutants to wild-type Arabidopsis (Columbia-0) plants, with the crosses using heterozygous mutant plants as the pollen donor expectation that segregation would occur in a Mendelian ratio of (wild type 3 CBR1/cbr1), the cbr1 allele was transmitted at very

Table 1. Segregation Analysis of the cbr1 Mutant Alleles in Progeny From Self-Pollinated cbr1 Heterozygotes

Genotype from PCR Analysis

CBR1/cbr1-2 % Carrying T-DNA cbr1/cbr1 CBR1/cbr1 CBR1/CBR1 P Valuea

T2 plants 43% 0 22 29 <0.0001 T3 plants 55% 6 52 48 <0.0001 CBR1/cbr1-3 T2 plants 62% 0 18 11 T3 plants 42% 0 45 62 <0.0001 T4 plants 57% 0 20 15 0.0011 CBR1/cbr1-4 T2 plants 63% 0 19 11 0.0061 T3 plants 49% 0 19 20 <0.0001 T4 plants 53% 0 16 14 0.0014 Heterozygous cbr1 plants were allowed to self-pollinate for each generation. The observed segregation ratios of the progeny were compared with the expected Mendelian ratio of 1:2:1; P values were calculated using the x2 test, 2 df. aSignificant differences between the observed ratio and the expected ratio 1:2:1. CBR1 and Male Gametophyte Function 3055

low rates. Most of the progeny were wild type in these test crosses: 24 of 24 were wild type for the CBR1/cbr1-2 testcross, 26 of 28 for the CBR1/cbr1-3 testcross, and 15 of 18 were wild type for the CBR1/cbr1-4 testcross. The low transmission of all three cbr-1 alleles indicates that the male gametophytes of the cbr1 mutants are substantially nonfunctional. We later genotyped, by PCR, 283 F2 progeny of two self-pollinated heterozygous plants derived from the wild type 3 CBR1/cbr1-3 testcross and found no homozygotes. Similar results were observed for the wild type 3 CBR1/cbr1-4 testcross, with no homozygous mutants identified among progeny of heterozygotes. Because there was some transmission of cbr1 alleles through the pollen, the testcross results indicate that a small proportion of cbr1 pollen is functional, and we infer that zygotes homozygous for cbr1-3 and cbr1-4 insertion al- leles did not develop into viable embryos. Even though our test- crosses to the wild type indicated low viability of cbr1-2 pollen, homozygous cbr1-2 mutants were identified as viable plants.

Initial Characterization of the Homozygous cbr1-2 Mutant

The viable cbr1-2 homozygotes obtained allowed us to carry out phenotypic and biochemical analyses. First, homozygous cbr1-2 plants were analyzed for the presence of CBR1 tran- script. Despite extensive RT-PCR analysis, no full-length tran- script was identified from homozygous cbr1-2 (Figure 1C), even after a secondary nested PCR of the RT-PCR product (see Supplemental Figure 1A online). Additional RT-PCR using pri- mers specific to the region of cbr1-2 59 to the T-DNA insertion site also failed to detect any RNA (see Supplemental Figure 1B online). However, RT-PCR produced a product corresponding to CBR1 sequence 39 of the T-DNA insertion (Figure 1C). To de- termine if part of the T-DNA insert was transcribed with this CBR1 mRNA, we designed primers (see Supplemental Table 1 online) to amplify from within the T-DNA left border to the 39 end of CBR1. RT-PCR indicated that amplification was possible more than 1300 bp into the T-DNA insertion (see Supplemental Figure 1B online). We consider it unlikely that this artifactual transcript could be translated into a functional protein. The transcript has stop codons in all three reading frames, including one in frame with the CBR1 coding sequence 105 bp upstream of the first preserved codon in the CBR1 open reading frame. The cbr1-2 insertions disrupt the CBR1 reading frame at residue 92, and based upon the three-dimensional crystal structure of human CBR1, the mutation removes four segments of the six-region Greek-key b-barrel that forms the FAD binding domain (Bando et al., 2004). Mutation in this critical domain Figure 2. most likely eliminates or severely compromises activity of any Characterization of Homozygous cbr1-2 Mature Seeds. protein produced. (A) Mature seeds of the wild type (WT) (left) and cbr1-2 (right). Mature cbr1-2 seeds were shrunken and shriveled compared (B) Average seed weight. with wild-type seeds (Figure 2A), and quantitative measure- (C) Total fatty acid methyl esters (FAME) per seed. ments confirmed their deficiencies. Both average seed weight (D) Fatty acid composition of mature seeds of the wild type and cbr1-2. 6 SE (B) (C) (Figure 2B) and oil per seed, measured as total fatty acid methyl Data are mean for 18 replicates of 20 seeds each for and and mean 6 SE for three replicates for (D). esters (Figure 2C), were significantly less than the wild type. In [See online article for color version of this figure.] sum, oil per seed weight was reduced from 359 6 13 mgmg21 in the wild type to 281 6 20 mgmg21 for cbr1-2 seeds. To in- vestigate the possible developmental basis of the morpho- logically distorted seeds, we examined embryo development from 2 to 7 d after flowering using differential interference 3056 The Plant Cell

contrast microscopy. Up to the late torpedo stage of embryo When seeds were sown on agar medium supplemented with 1% development, we observed no visible differences between Suc, ;60% of cbr1-2 seeds germinated, compared with 98% of wild-type and cbr1-2 embryos (Figures 3A to 3F). However, wild-type seeds. All of the germinated cbr1-2 seeds produced while wild-type embryos consistently proceeded to the folded- a radicle >3 mm in length and were scored as established cotyledon stage (Figure 3G), the development of cbr1-2 seedlings (Figure 4A). Seeds of the cbr1-2 mutant also exhibited embryos was very variable. Some embryos folded like the delayed germination, with the median time to germinate on su- wild type, as shown in Figure 3H, while others folded in- crose medium >1.5 d compared with 0.6 d for wild-type controls completely (Figure 3I) or did not fold (Figure 3J). These ob- (Figure 4B). The success of seedling establishment was highly servations are consistent with the morphology of cbr1-2 variable, even after an extended, 7-d stratification regime. As seeds shown in Figure 2A and indicate that the deficiency in shown in Figure 5A, compared with wild-type controls, cbr1-2 cbr1-2 plants affects the late stages of embryo and seed seedlings were delayed in root and leaf development after 7 d of development. growth. Nevertheless, after 10 d of growth on Suc medium, In cbr1-2 seed, the proportion of 18:3 was significantly re- ;55% of cbr1-2 seedlings had expanded cotyledons and de- duced. For wild-type Arabidopsis seed, 18:3 is an abundant fatty veloping leaves. When these established seedlings were trans- acid, making up 21% of the total fatty acids, but cbr1-2 seed ferred to soil, the cbr1-2 plants varied in size, with most having had only 6% 18:3 (Figure 2D), and there was a concomitant smaller leaves and rosettes than wild-type controls (Figures 5B increase in 18:1 in the mutant. Calculations from the data in and 5C). The small proportion of cbr1-2 that germinated quickly Figure 2D indicate that 18:1 desaturation was reduced by 25% and developed in synchrony with wild-type seedlings grew into and 18:2 desaturation by 62% in cbr1-2 seeds, relative to wild- plants whose appearance and height were close to the wild type type controls. These changes are only slightly greater in mag- (Figure 5D), indicating that much of the size variation observed in nitude than those seen in the hypomorphic mutant cbr1-1 cbr1-2 plants was due to delayed germination and poor seedling (Kumar et al., 2006) and suggest a differential effect of reduced establishment. To test whether the cb1-2 mutation caused electron supply on activities of the FAD2 and FAD3 enzymes. a detectable change in desaturation or hydroxylation of any in- Analyses of individual phospholipids from seeds also showed dividual glycerolipid or sphingolipid, we conducted lipidomics decreases in 18:3 (see Supplemental Figure 2 online), reflecting analyses on wild-type and cbr1-2 leaf extracts. No statistically the fact that phosphatidylcholine is the substrate for fatty acid significant difference in composition was detected in any of the desaturation. Changes in fatty acid composition were confined 13 glycerolipid and sphingolipid components analyzed, and the to the seeds; there was no substantial change in fatty acid quantities of these individual lipids in cbr1-2 were entirely com- composition in the leaves or roots of cbr1-2 plants (see parable to those in the wild type (see Supplemental Figures 3 and Supplemental Table 2 online). 4 online). Homozygous cbr1-2 seeds germinated poorly on soil and the resulting seedlings did not establish and grow into mature Complementation of cbr1 Mutants plants. As shown in Figure 4A, on agar medium in the absence of Suc, some cbr1-2 seeds germinated (scored by radicle pro- To confirm that the phenotypic defects observed in cbr1 mutant trusion), but did not produce a radicle greater than 1 mm in plants are the result of a cytochrome b5 reductase defect, we length even after 8 d, at which time ;90% of the wild-type cloned a 3-kb genomic fragment containing the entire wild-type controls were established seedlings with substantial roots. CBR1 gene into a plant expression vector. Transformation of

Figure 3. Embryo Development in Wild-Type and cbr1-2 Seeds.

(A) to (F) From the heart to late-torpedo stage, wild-type (top row) and cbr1-2 (bottom row) embryos are similar in appearance. (G) to (J) The folded cotyledons visible in wild-type embryos (G) are seen in only some cbr1-2 embryos (H); other cbr1-2 embryos fold poorly (I) or not at all (J). CBR1 and Male Gametophyte Function 3057

Homozygous cbr1-2 Plants Have Low Seed Set and Outcross More Readily Than the Wild Type

While homozygous cbr1-2 plants were often, but not always, smaller than the wild type, the most striking visual phenotype of mature plants was very low seed set. Even on the largest, robust cbr1-2 plants, siliques were no greater than half the length of those of wild-type plants (Figure 6A). Dissection of these small siliques revealed very few seeds in each silique. Most of the ovules were white and shriveled, suggesting that they were unfertilized (Figure 6B); only two or three developing seeds were found in each cbr1-2 silique at 9 to 10 d after pollination (Figure 6C). The total number of ovules formed (fertilized + unfertilized) in cbr1-2 siliques was also reduced from wild-type numbers. During early characterization of homozygous cbr1-2 plants, we observed that heterozygous cbr1-2 plants routinely occurred in seed stocks derived from parents identified as homozygous. To test the hypothesis that cbr1-2 plants outcross more readily than wild-type Arabidopsis, we interplanted homozygous cbr1-2 with plants homozygous for a transgenic Discosoma Red (DsRed) marker. Progeny seeds of this DsRed transgenic line can be identified by their fluorescence (Stuitje et al., 2003). For comparison, wild-type plants were similarly interplanted with the same DsRed transgenic line. Before the siliques began to sen- Figure 4. Germination and Establishment of cbr1-2. esce, the plants expressing DsRed were removed to eliminate (A) Germination (radicle protrusion) and establishment for wild-type (WT) their seed as contaminants, and the cbr1-2 and wild-type plants or homozygous cbr1-2 seeds on agar media with or without 1% Suc. were individually tied. At maturity, wild-type and cbr1-2 plants Establishment was scored as root length >3 mm at 8 d. Data are mean 6 were harvested and red fluorescent seeds from each plant SE for two experiments with 70 to 80 seeds per treatment. counted. Red fluorescent seeds derived from the cbr1-2 line (B) Time course of germination (radicle protrusion) on agar media with were germinated on Basta-supplemented medium to confirm 1% Suc. Data are mean 6 SE for four experiments with 20 to 35 seeds presence of the T-DNA insertion; seeds that were both red and per replicate. herbicide resistant were progeny of cbr1-2 plants produced by outcrossing. We found a 23-fold increase in the rate of out- homozygous cbr1-2 plants with this CBR1 genomic clone re- crossing for cbr1-2 compared with the wild-type control plants; stored both 18:3 and 18:1 fatty acids in T2 seed to wild-type almost 1% of the cbr1-2 seed resulted from outcrossing events levels (see Supplemental Table 3 online). Germination and (Figure 7). These data further demonstrate the reduced trans- seedling growth of the resulting cbr1-2 CBR1 T2 transgenic mission of the cbr1-2 allele. plants were also restored by the complementation transgene and were similar to the wild type. Almost all of these cbr1-2 plants contained the complementation transgene, since trans- Reduced Fertilization in cbr1-2 Is Caused by Poor Pollen mission of the cbr1-2 allele was very low in noncomplemented Tube Growth pollen and noncomplemented homozygous cbr1-2 seeds ger- minated at a low rate. This complementation was equally suc- The combined phenotypes of reduced seed set, high rate of cessful for the other insertion alleles. When plants heterozygous cross-pollination, and reduced transmission via the male ga- for the cbr1-3 mutation were transformed with the CBR1 ge- metophyte led us to examine pollen viability and function in nomic clone, it was possible using PCR to identify, among the cbr1-2 plants. We used Alexander’s stain (Alexander, 1969) to self-pollinated progeny of the transformants, plants homozy- assess pollen viability. On the basis of this test, the pollen of gous for the cbr1-3 mutation, which never occurred in the un- cbr1-2 was 99% 61% (n = 5) viable, comparable to the 98% 6 transformed mutant line. All homozygous plants contained the 3% (n = 5) viability of wild-type pollen. Pollen of the mutant also CBR1 transgene, and no homozygous cbr1-3 plants were appeared normal in size and shape (Figure 8A). identified that lacked the CBR1 transgene (see Supplemental To assess in vivo pollen germination and pollen tube growth, Table 4 online). To complement the third insertion mutant, het- we used aniline blue stain to visualize pollen tubes within the erozygous cbr1-4 plants were crossed with a complemented stigma and style (Mori et al., 2006). In self-pollinated wild-type cbr1-3/CBR1 line. As observed for the other mutants, cbr1-4 flowers, pollen germinated and pollen tubes grew through the homozygotes were obtained when, and only when, the line transmitting tract, reaching the ovules. However, in self- contained the CBR1 transgene (see Supplemental Table 4 on- pollinated cbr1-2 flowers, a substantial portion of the pollen line). Complementation of all three cbr1 insertion mutants by the germinated but most of the pollen tubes stopped growing before CBR1 genomic clone demonstrates that the phenotypes de- reaching the ovules (Figure 8B). For approximately two events scribed result from mutations within the CBR1 gene. per pistil, a cbr1-2 pollen tube grew through the transmitting 3058 The Plant Cell

Figure 5. Phenotypic Characterization cbr1-2 Plants. (A) Seedlings of wild-type (WT) and cbr1-2 grown together for 7 d on agar media with 1% Suc. Bars = 1 mm. (B) Plants transferred to soil at 10 d and grown for an additional 11 d on soil. (C) Leaves removed from one wild-type and one cbr1-2 plant shown in (B). (D) Mature wild-type and cbr1-2 plants showing the slightly reduced size of mutant plants.

tract and reached an ovule, a low success rate of pollen tube pistils (wild type 3 cbr1-2), pollen apparently germinated but did growth that correlates well with the observed seed set of cbr1-2 not extend normal pollen tubes (Figure 9A). When assessed at plants (Figure 6). In germination tests on artificial media (Boavida 9 d after pollination, wild type 3 cbr1-2 seed set (Figures 9B and and McCormick, 2007), only 3% of cbr1-2 pollen grains pro- 9C) was similar to the seed set from cbr1-2 self-pollinated sili- duced a pollen tube >20 mm long. Therefore, attenuated pollen ques (Figure 6). These results show that the dysfunctional pollen tube growth in cbr1-2 is principally responsible for low seed tube growth of cbr1-2 is substantially independent of the female production in the mutant line. gametophyte and that the critical requirement for CBR1 is in To confirm that the CBR1 gene was actively transcribed in pollen tube growth. pollen and pollen tubes, we fused the CBR1 promoter to the b-glucuronidase (GUS) reporter (Curtis and Grossniklaus, 2003) and transformed wild-type Arabidopsis plants with the con- DISCUSSION struct. When pollen from T1 plants was germinated in vitro (Boavida and McCormick, 2007) and stained for GUS activity, it Biochemical Compensation for the Loss of CBR1 was readily apparent that the CBR1 promoter was active in both hydrated pollen and growing pollen tubes (Figure 8C), consistent Cytochrome b5 reductase transfers electrons from NADH to with results from transcriptional profiling experiments (Wang cytochrome b5 proteins, which then transfer the electrons to et al., 2008). fatty acid desaturases (Shanklin and Cahoon, 1998), hydroxy- Pollen tube guidance and fertilization can be disrupted by lases (Nagano et al., 2009), and other enzymes. While not yet mutations affecting sporophytic tissues, such as the trans- characterized biochemically, enzymes with N-terminal cytochrome mitting tract or embryo sac, or by the female gametophyte, so b5 domains that incorporate a characteristic heme binding motif even though our testcrosses indicate a defective male game- most likely acquire electrons via CBR1 and cytochrome b5, as has tophyte, the results do not preclude an additional contribution been established in the family of desaturases with similar cyto- from the maternal tissue or female gametophyte. To test this chrome b5 domains (Sperling et al., 1998; Gostincar et al., 2010). possibility, we conducted reciprocal testcrosses between the There is considerable evidence that NADPH:cytochrome wild type and cbr1-2 and stained the pistils 24 h after pollination P450 reductase, whose role is to transfer electrons from NADPH with aniline blue to assess pollen tube growth. When wild-type to the cytochrome P450 monooxygenases, can also supply pollen was used as the pollen donor for either wild-type pistils electrons to the cytochrome b5 pathway. In vitro analysis of (wild type 3 wild type) or mutant pistils (cbr1-2 3 wild type), mammalian electron transfer demonstrates that the P450 re- pollen tube growth was prolific throughout the transmitting tract ductase can supply electrons to cytochrome b5 for fatty acid (Figure 9A). By contrast, when cbr1-2 pollen was used as the desaturation (Enoch and Strittmatter, 1979; Dailey and Strittmatter, pollen donor for either mutant (cbr1-2 3 cbr1-2) or wild-type 1980). In addition, P450 reductase supports or enhances many CBR1 and Male Gametophyte Function 3059

However, our examination of cbr1 insertional mutants shows that loss of cytochrome b5 reductase disrupts reproduction, demonstrating the essential nature of cytochrome b5 reductase in Arabidopsis. We found that all three T-DNA insertional mutants examined in CBR1 were male gametophyte defective (Table 1). No homo- zygous progeny of cbr1-3 or cbr1-4 could be isolated, and while we were able to identify homozygous progeny of heterozygous cbr1-2 plants, they were present at only 6% of the total progeny, substantially below the 25% frequency expected from Mende- lian segregation (Table 1). When we fertilized wild-type plants with pollen from lines heterozygous for either cbr1-3 or cbr1-4, a low proportion of the progeny were heterozygous for the cbr1 allele, so some male gametophytes in these two lines are viable. There is a possibility that either cbr1-3 or cbr1-4 heterozygotes could segregate homozygous progeny, albeit at much lower rates than cbr1-2, since our limit of detection was <0.1%. No full-length CBR1 transcript was detected in homozygous cbr1-2 plants, even when analyzed by nested PCR amplification (see Supplemental Figure 1 online). The stable partial transcript that we detected (Figure 1; see Supplemental Figure 1 online) is unlikely to encode a functional protein, although this possibility cannot be excluded. The complementation of cbr1-2, cbr1-3, Figure 6. Characterization of cbr1-2 Siliques and Seed Set. and cbr1-4 by a CBR1 genomic clone (see Supplemental Tables (A) Siliques of the wild type (WT) and cbr1-2. 3 and 4 online) demonstrates that the described mutant phe- (B) Dissected siliques of the wild type and cbr1-2 showing reduced seed notypes represent loss of function of the CBR1 gene. Isolation of set in the mutant. homozygous cbr1-2 plants (Figure 1B) permitted further analysis (C) Fertilized ovules (green seeds) and unfertilized (white and shriveled) of the function of CBR1 in Arabidopsis. ovules were counted from dissected siliques depicted in (B). Data are 6 mean SE for 15 siliques. CBR1 Is Substantially Redundant during Vegetative Growth [See online article for color version of this figure.] CBR1 is an integral membrane protein of the ER that provides reductant to desaturases and other enzymes via cytochrome b5 cytochrome b5 activities (Guengerich, 2005). Mice deleted in (Fukuchi-Mizutani et al., 1999). According to immunolocalization cytochrome b5 are viable, but greatly perturbed in their P450 in BY-2 cells, Arabidopsis CBR1 localizes to the ER membrane metabolism, demonstrating in vivo the interconnection between (Shockey et al., 2005) and has been identified in the ER the two electron transfer pathways (Finn et al., 2011). Indeed, in S. cerevisiae, cytochrome b5 reductase activity is completely dispensable, while simultaneous loss of cytochrome b5 reductase and P450 reductase is lethal (Tiedje et al., 2007). Compensation for loss of cytochrome b5 reductase is not always so complete. Human methemoglobinemias are genetic diseases caused by mutations affecting cytochrome b5 electron transfer. In milder forms of the disease, these mutations produce treatable cyanosis (Hultquist et al., 1993), but in severe cases disruption in fatty acid and cholesterol metabolism damages neurological development, resulting in death during early child- hood (Percy et al., 2005). Clearly, NADPH:cytochrome P450 reductase is incapable of fulfilling all roles of CBR1 in humans. In plants, recombinant Arabidopsis P450 reductase proteins have been shown to supply electrons to cytochrome b5 proteins in vitro (Fukuchi-Mizutani et al., 1999), suggesting at minimum Figure 7. Outcrossing Frequency for Wild-Type and cbr1-2 Plants. that support for cytochrome b5 activity is possible through the The wild type (WT) and mutant were interplanted with transgenic plants P450 reductase electron transfer pathway. An Arabidopsis mu- homozygous for the DsRed fluorescent marker. The numbers of fluo- tant with greatly reduced cytochrome b5 reductase activity ex- rescent seeds and total seeds per plant were counted at harvest. hibited no phenotypic change other than reduction of 18:3 fatty Presence of the cbr1-2 insertion was confirmed by Basta selection of acids in mature seeds (Kumar et al., 2006), demonstrating that fluorescent seed derived from that line. Data are mean 6 SE for eight much of the cytochrome b5 reductase activity is dispensable. plants. 3060 The Plant Cell

electron transfer through the two Arabidopsis isoforms of this contribute to this compensation. Both Arabidopsis P450 reductase enzymes are located in the ER membrane and readily assayed in microsomal fractions (Mizutani and Ohta, 1998; Fukuchi-Mizutani et al., 1999). Previous research established that microsomal preparations of plant tissues can use NADPH to reduce cytochrome b5–dependent enzymes, including desa- turases, presumably as a result of cytochrome P450 reductase activity (Slack et al., 1976; Smith et al., 1990). Furthermore, re- combinant Arabidopsis P450 reductase proteins are able to supply electrons to cytochrome b5 in vitro (Fukuchi-Mizutani et al., 1999).

CBR1 Is Necessary for Proper Seed Maturation and Germination

The observed decrease of 18:3 and increase of 18:1 in the mature seed of cbr1-2 plants (Figure 2D) was not itself surpris- ing, since CBR1 supplies electrons via cytochrome b5 to the FAD2 and FAD3 desaturases responsible for synthesis of 18:3 (Smith et al., 1990; Browse et al., 1993). These findings conform to analysis of the hypomorphic cbr1-1 mutant (Kumar et al., 2006). Since the fatty acid compositions of cbr1-2 leaves and roots were similar to the wild type (see Supplemental Table 2 online), we suspect that the low 18:3 in the seeds is due to the high demand for reductant imposed by fatty acid desaturation during oil accumulation in developing seeds; the lower rates of fatty acid synthesis in the leaf and root tissue permit their composition to remain unaffected. In photosynthetic tissue, the Figure 8. Pollen Tube Growth Is Reduced in the cbr1-2 Mutant. plastidial desaturases FAD7 and FAD8, which use ferredoxin as fl (A) Both wild-type (WT) and cbr1-2 pollen grains are identified as viable reductant, can also compensate for the reduced ux through the by Alexander’s stain. ER desaturases (Browse et al., 1993). In any tissue, reductant (B) In vivo pollen germination of the wild type shows robust pollen tube may also come via NADPH:P450 reductase isozymes; the mini- growth, while few pollen tubes are seen extending beyond the stigma in mal changes in root fatty acid composition in particular support cbr1-2. the hypothesis that CBR1 is substantially redundant with P450 (C) Expression of a CBR1pro:GUS reporter in wild-type pollen. Pollen reductase proteins in vegetative tissues. grains were hydrated in water (left) or germinated (right) for 16 h on solid Although the requirement of cbr1-2 seeds for an exogenous germination medium before staining for GUS activity. carbon source to germinate (Figure 5A) is reminiscent of the Suc-dependent class of mutants that either fail to hydrolyze fatty proteome using isotope tagging and liquid chromatography– acids from triacylglycerol or fail to breakdown the fatty acids via tandem mass spectrometry (Heazlewood et al., 2004; Dunkley b-oxidation in the peroxisome (Hayashi et al., 1998; Fulda et al., et al., 2006). Recently, a case has been made that CBR1 also 2004; Eastmond, 2006), it is more likely that the low germination localizes to the outer mitochondrial membrane (Duncan et al., and establishment rates were caused by a decrease in total fatty 2011). A second isozyme of cytochrome b5 reductase, CBR2, is acid content in cbr1-2 seeds (Figure 2) or by a deficiency in 38% identical to the CBR1 protein sequence and found in the a CBR1-related function distinct from triacylglycerol metabo- mitochondrial proteome, likely localized to the inner mitochon- lism. The reduced oil, shriveled seeds, and delayed germination drial membrane (Heazlewood et al., 2004). While the function of of cbr1-2 seeds (Figures 2 and 4) are similar to the seed phe- this isoform is unknown, our mutant analysis clearly shows that notypes of the tag1 mutant, deficient in diacylglycerol acyl- CBR2 is incapable of meeting the need for reductant in the ER. transferase activity (Routaboul et al., 1999). However, the delayed The vegetative tissues of cbr1-2 are not strongly affected by germination of tag1 seeds is not affected by Suc, and tag1 seeds the mutation. The variability in size of cbr1-2 plants (Figure 5) showed >98% germination after 9 d of incubation on medium was most likely a result of delayed germination and poor with or without Suc. Nevertheless, we consider it likely that the seedling establishment (Figure 5B) that results from deficiencies reduced oil of cbr1-2 seeds contributes, in some degree, to the evident in mature cbr1-2 seeds (Figure 2). The cbr1-2 plants that germination and seedling establishment phenotypes. germinated early and established well were equivalent to the wild type in vegetative growth and development, indicating that CBR1 Is Required for Function of the Male Gametophyte other pathways for electron supply effectively compensate for the loss of CBR1 activity during much of the plant lifecycle. It is Segregation of the cbr1 insertional mutants at ratios of 1:1 likely, given the role of P450 reductase in other organisms, that (heterozygous:wild type) (Table 1) indicated a gametophytic CBR1 and Male Gametophyte Function 3061

Figure 9. Reciprocal Crosses Illustrate the Gametophytic Defect in cbr1-2.

(A) Aniline blue staining of manual crosses, 24 h after pollination. WT, the wild type. (B) Siliques from manual crosses, 9 d after pollination. (C) Seeds per silique from manual crosses (n = 8); error bars indicate SE. Crosses labeled as female 3 male in (A) to (C). defect, and reciprocal backcrosses of heterozygous cbr1 mu- pollen, when pollen of cbr1-2 plants contacted the stigmas of tant lines with the wild type pinpointed the defect in the male wild-type or mutant plants, germination appeared robust, and gametophyte. At this critical stage of the plant life cycle, com- pollen tubes began extension into the stigma tissue, but ceased pensation for loss of CBR1 activity is evidently insufficient to shortly thereafter. maintain cellular function. The gametophytic defect cannot be For pollen of cbr1-2 plants, only rarely did pollen tubes pen- solely the result of reduced 18:3 fatty acid, since Arabidopsis etrate the style and proceed along the transmitting tract to effect fad3 mutant plants have greatly reduced 18:3 but produce normal fertilization, whether these pollen were transferred naturally gametophytes (Browse et al., 1993), so the observed defect must be (Figure 8) or by hand (Figure 9). This pattern bears some simi- due to the loss of CBR1 electron supply to enzymes other than the larity to the male reproductive defect seen in plants mutant in fatty acid desaturases. The enzymes affected by CBR1 deficiency the pectin methylesterase homolog VANGUARD1, whose pollen might even be P450 monooxygenases, since cytochrome b5 has tubes penetrate the style much more slowly than wild-type been shown to contribute electrons to P450 39,59-hydroxylases tubes (Jiang et al., 2005). Pollen from pip5k4 mutants, defective involved in flavonoid metabolism in petunia (Petunia hybrida;de in phosphatidylinositol-4-monophosphate 5-kinase4, produce Vetten et al., 1999) and grape (Vitis vinifera; Bogs et al., 2006). pollen tubes that take about twice as long as the wild type to The male gametophyte defect revealed by segregation anal- achieve fertilization (Sousa et al., 2008). Defects like these that ysis of all three insertion mutants, coupled with the low seed set slow pollen tube growth often result in seed predominately at of the sole insertion line that produced homozygous plants, led the top of the silique (Muralla et al., 2011), a pattern not dis- us to examine pollen function in cbr1-2 plants. Alexander’s stain cernible in cbr1-2 mutants, whose few seeds appeared ran- indicated that viability of cbr1-2 pollen grains at anthesis was domly distributed throughout the length of the silique (Figure 6). comparable to the wild type (Figure 8A), and when cbr1-2 pollen Some mutations causing slow pollen tube growth fail to exit the was applied to either cbr1-2 or wild-type anthers, many pollen transmitting tract and target ovules for fertilization. Arabidopsis grains germinated. However, nearly all pollen tube growth plants mutant in TFIIB1, a general transcription factor (Zhou ceased soon after germination and only a very few pollen tubes et al., 2013), or mutant in a homolog to the animal translationally grew through the transmitting tract to complete fertilization controlled tumor protein (Berkowitz et al., 2008), both show (Figures 8 and 9). The process of fertilization in Arabidopsis defects in targeting ovules; however, no such defect is evident in unfolds in stages after a pollen grain alights on a papillary cell of the cbr1-2 mutant, which successfully fertilized ovules when the the stigma (Palanivelu and Tsukamoto, 2012). Like wild-type pollen tubes traversed the transmitting tract (Figures 8 and 9). 3062 The Plant Cell

Is a Single Cytochrome b5–Dependent Enzyme Responsible it is possible that greatly decreased activity of several or all of for the cbr1-2 Phenotype? these enzymes provide combined effects on membrane structure and function that result in the defect in pollen tube growth we Our lipid analyses of vegetative tissue of the cbr1-2 mutant observed. Alternatively, we cannot exclude the possibility that – indicate that the many cytochrome b5 dependent reactions an as yet uncharacterized, cytochrome b5–dependent enzyme is required for the synthesis of membrane glycerolipids and responsible. Nevertheless, the relatively normal vegetative growth sphingolipids are maintained, presumably because the ATR1 of cbr1-2 plants supports the notion that sphingoid base hy- and ATR2 NADPH:P450 reductase enzymes maintain adequate droxylase as well as other cytochrome b5–dependent enzymes reduction of the cytochrome b5 pool. The low 18:3 found in the are provided electrons through the ability of NADPH:P450 re- seed oil and seed phospholipids, together with the seed phe- ductase to reduce cytochrome b5 (Fukuchi-Mizutani et al., 1999). notypes we observed, indicate that cytochrome b5 reduction is We conclude that the deficiency of cytochrome b5 reductase is partially compromised during seed development. However, it is masked in most tissues and organs of the cbr1-2 mutant by the the defect in pollen of cbr1-2 (Figures 8 and 9), cbr1-3,andcbr1-4 activity of NADPH:P450 reductase encoded by ATR1 and ATR2. (Table 1) that is the key failure in the lifecycle of the mutants. We In pollen, and to a lesser extent in developing seeds, the P450 therefore considered whether electron supply from cytochrome reductase isozymes apparently fail to adequately maintain re- b5 to a single, critical enzyme of membrane lipid synthesis might duction of the cytochrome b5 pool, resulting in a pleiotropic effect cause the defect in pollen tube growth observed in cbr1-2. on cytochrome b5 enzymes. In glycerolipid metabolism, the major cytochrome b5– dependent reactions are those performed by the endoplasmic METHODS reticulum 18:3 desaturase, FAD3, and the 18:2 desaturase, FAD2. However, the fad3 and fad2 mutants both produce functional pollen (Miquel and Browse, 1992; Browse, et al., 1993), in- Plant Growth Conditions dicating that an effect of the cbr1-2 mutation on either of these Arabidopsis thaliana (ecotype Columbia-0) wild-type and mutant plants enzyme activities cannot, by itself, explain the pollen defect. The were cold-stratified for 2 d at 4°C, unless otherwise specified, and ger- sphingolipid D4desaturase (At4g04930) is highly expressed in minated on 13 Murashige and Skoog medium (Sigma-Aldrich) supple- flowers and pollen, but characterization of two knockout in- mented with 1% (w/v) Suc and 0.75% agar, under 16 h day/8 h night at m 22 21 sertion mutants showed no difference in pollen viability, relative 22°C, with a 80 mol m s light intensity. Seedlings 10 to 12 d old were transferred to soil (Pro-Mix; Premier Tech Horticulture) and grown under to the wild type (Michaelson et al., 2009). The sphingolipid D8 24-h light at 22°C with 200 mmol m22 s21 light at rosette height. Re- desaturase is encoded by two , sld1 and sld2, but double sistance to glufosinate (Basta; Bayer) was determined by watering plants D mutants with no detectable 8 desaturation reproduce normally twice prior to bolting, 3 to 4 d apart, with 0.011% Basta. and have relatively mild phenotypes (Chen et al., 2012). Simi- larly, mutants defective in the ACYL DESATURASE2 (ADS2) Identification of cbr1 Mutants gene encoding 24:0/26:0 n-9 desaturase (Smith et al., 2013) reproduce normally and exhibit a vegetative phenotype only at The insertion site for SALK_129878 is immediately upstream of the CBR1 open reading frame and lines homozygous for the insertion expressed the low temperatures (Chen and Thelen, 2013). The FATTY ACID full-length CBR1 cDNA. The insertion site for sail_579_A02 is reportedly in HYDROXYLASE1 (FAH1) and FAH2 genes encode enzymes that the first intron of the CBR1 gene, but no insertion was detectable under a -hydroxylate the fatty acids on sphingolipids (König et al., standard PCR conditions. These two insertions were not further analyzed. 2012; Nagano et al., 2012). fah1 fah2 double mutant plants show The T-DNA insertional lines SAIL_644_A11 (Sessions et al., 2002) and moderately reduced growth and other phenotypes but no in- WiscDsLox 377-380021 (Woody et al., 2007) were obtained from the dication of an effect on reproduction (König et al., 2012). A ca- ABRC at Ohio State University, and Gabi-Kat Line 371A08 was obtained veat to this observation is that the fah1 allele used in the cross is from Professor Bernd Weisshaar Gabi-Kat at Universität Bielefeld, Ger- severe but not completely null. The sphingoid base hydroxylase1 many (Rosso et al., 2003). Plants were genotyped by PCR using separate (sbh1) sbh2 double mutants lacking sphingoid base hydroxylase reactions with three primers, left border of the T-DNA insertion of cbr1 (primers LB3, WiscLB, or GKLB; see Supplemental Table 1 online) and do have strong phenotypes, with homozygous double mutant a CBR1 gene-specific primer pair (primers P1 and P2 for cbr1-2 and cbr1-4 seedlings showing very slow growth and a failure to progress and P3 and P4 for cbr1-3; see Supplemental Table 1 online). from vegetative to reproductive growth (Chen et al., 2008). To test for transcript in cbr1-2, RT-PCR was performed by isolating However, the double mutants were obtained in the expected RNA with Trizol reagent (Life Technologies) from leaf and inflorescence, Mendelian ration from a SBH1/sbh1 SBH2/sbh2 parent, in- followed by DNaseI treatment with the DNA-free RNA kit (Zymo Research). dicating that sbh1/sbh2 pollen had no significant deficiency cDNA was made using the Superscript III first-strand synthesis system (Life relative to wild-type or SBH1 SBH2 (or SBH1 sbh2 or sbh1 Technologies) and amplified with designated primers for 30 cycles of PCR. SBH2) pollen produced by the parent plant. Other pathways of When Gabi-Kat line 371A08 seeds were sown on Murashige and – cellular biochemistry are not so completely defined, but we Skoog agar supplemented with 1% (w/v) Suc and sulfadiazine, the seedlings were 98% resistant; the infrequent occurrence of wild-type could find no reports of any cytochrome b5–dependent enzyme plants indicates three unlinked insertion sites. A heterozygous cbr1-4 in Arabidopsis or other plant species for which a gene mutation plant was backcrossed into wild-type Arabidopsis (Columbia-0), and F2 has been shown to confer a male gametophyte lethal phenotype CBR1/cbr1-4 plants from this backcross were self-pollinated. A line (Muralla, et al., 2011). chosen for further analysis produced progeny that were 1:1 wild type: While deficiencies in the individual cytochrome b5–dependent sulfadiazine resistant; these resistant plants were found to be hetero- enzymes discussed here apparently do not affect pollen function, zygous for the cbr1-4 allele. CBR1 and Male Gametophyte Function 3063

Lipid Analysis scored and divided by the number of seeds per plant as estimated by seed weight. To ensure that the outcrossing represented events in cbr1-2 Fatty acids of mature seeds (20 to 50 seeds) were derivatized to fatty acid plants, DsRed seeds harvested from cbr1-2 plants were germinated on methyl esters in 1 mL of 2.5% (v/v) sulfuric acid in methanol for 1.5 h at medium containing 20 mg/mL glufosinate-ammonium (Basta). 80°C (Miquel and Browse, 1992). Fatty acid methyl esters were extracted into hexane and analyzed by gas chromatography with flame ionization detection on a wax column (EC Wax; 30 m 3 0.53 mm i.d. x 1.20 mm; Pollen Analysis Alltech) with parameters as follows: 220°C for 2 min followed by a ramp to Pollen viability was assayed using Alexander’s staining (Alexander, 1969) 245°C at 10°C min21, with a 4-min final temperature hold. Total seed oil and visualized on a Leica DM2000 microscope and images captured with was quantified according to Li et al. (2006), except 20 seeds were used per a Leica DFC295 camera. Pollen was germinated in vitro by the method of sample instead of 50 seeds. Boavida and McCormick (2007). Pollen tubes growing in vivo were stained Lipids were extracted from leaf tissues according to Welti et al. (2002). with aniline blue following sample preparation (Mori et al., 2006); the Total lipids from mature seeds were extracted following the protocol from aniline blue staining solution was 0.1% aniline blue, 10 mM CHES buffer, the Kansas Lipidomics Research center (http://www.k- state.edu/lipid/ pH 10, and 100 mM KCl. Stained pistils were observed with a Leica lipidomics/). Seed lipids were fractionated on a silica column, eluting the DM LFS upright microscope equipped with water immersion lenses not neutral lipids with 5 mL of chloroform/methanol (99:1, v/v) and the polar corrected for cover slips (HCX Plan-APOU-V-I series) and images cap- lipids with 5 mL of chloroform/methanol/water (5:5:1, v/v/v), followed by tured with a Leica DFC 300 FX cooled charge-coupled device camera. 2 mL chloroform and 2 mL 0.88% KCl. Polar lipids were washed with 2 mL of chloroform, dried under nitrogen, and resuspended in 200 mLof chloroform. Thin layer chromatography was used to separate polar lipids, GUS Assay with double development in acetone/toluene/water (91:30:8, v/v/v) on K6 The CBR1 promoter was cloned into pENTR (Invitrogen) using primers Silica Gel 60A plates (Whatman) that were dipped in 0.15 M ammonium CBR1-PRM-topo and CBR1-PRM-end (see Supplemental Table 1 online) sulfate and dried before use. Separated lipids were stained with 0.005% and subcloned into plant transformation vector pMDC162 (Curtis and primuline in 80% acetone, visualized under UV light, and identified by Grossniklaus, 2003) upstream of the GUS gene with Clonase I (Invitrogen). comigration with standards. Phosphatidylcholine, phosphatidylethanol- The resulting construct was transformed into Agrobacterium strain amine, and phosphatidylinositol silica bands were collected and fatty acid GV3101 and transgenic plants generated by floral dip (Clough and Bent, methyl esters analyzed as described above, after addition of a known 1998). Pollen from T1 plants was hydrated in water or germinated in vitro amount of 15:0 internal standard. Total leaf lipids were separated using on solid medium (Boavida and McCormick, 2007) for 16 h before staining the same thin layer chromatography, and the major phospholipid and in GUS assay buffer (0.1 M sodium phosphate buffer, pH 7.2, 10 mM galactolipid bands analyzed as described. Methods for extraction, sep- EDTA, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, aration, and analysis of sphingolipids were as described by Markham and 1 mM X-glucuronide, and 0.1% [v/v] Triton X-100) for 24 h at 37°C. The Jaworski (2007). stained pollen was visualized as described above.

Complementation Cloning Accession Numbers For trans-complementation of cbr1 mutants with a genomic clone, an Sequence data from this article can be found in the Arabidopsis Genome ;3-kb fragment of genomic DNA containing the CBR1 promoter, CBR1 Initiative or GenBank/EMBL databases under the following accession gene, and CBR1 39untranslated region was amplified using primers with numbers: CBR1, AED92466.1, AT5G17770; CBR2, P83291, AT5G20080; NotI restriction sites (primers CBR1-PRM and CBR1-3UTR; see Supplemental CB5, AT1G26340, AT2G32720, AT2G46650, AT5G48810, AT5G53560, Table 1 online). This fragment was blunt-end cloned into pCRScript-Amp AT1G60660; ATR1, AT4G24520; ATR2, AT4G30210; FAD2, AT3G12120; (Agilent Technologies) following the manufacturer’s instructions and then FAD3, AT2G29980; D4-DES, AT4G04930; SLD1, AT3G61580; SLD2, digested with NotI and the genomic fragment cloned into the NotI site of plant AT2G46210; ADS2, AT2G31360; FAH1, AT2G34770; FAH2, AT4G20870; transformation vector pART27 (Gleave, 1992). The resulting construct was SBH1, AT1G69640; and SBH2, AT1G14290. transformed into Agrobacterium tumefaciens strain GV3101 and transgenic plants generated by floral dip (Clough and Bent, 1998). Supplemental Data

Differential Interference Contrast Microscopy The following materials are available in the online version of this article. Siliques at various stages of development were harvested, dissected, and Supplemental Figure 1. Further RT-PCR Analysis of Homozygous fixed for 48 h in ethanol/acetic acid (8:1). Specimens were cleared in cbr1-2. Hoyer’s solution (Liu and Meinke, 1998) for an additional 48 h, whole Supplemental Figure 2. Fatty Acid Compositions of Major Seed mounted, and observed using an Olympus microscope equipped with Phospholipids. Nomarsky differential interference contrast optics. Images were captured Supplemental Figure 3. Fatty Acid Compositions of Major Leaf using ProGress Image Analysis. Glycerolipids. Supplemental Figure 4. Compositional Analyses of the Long-Chain Quantification of Outcrossing Events Base and Fatty Acid Components of Sphingolipids from Leaves of the To quantify outcrossing events, cbr1-2 mutant plants were interplanted in Wild Type and cbr1-2. 10-cm pots (five plants per pot) with a transgenic Arabidopsis line known Supplemental Table 1. List of Primers. to be homozygous for the DsRed selection marker (Stuitje et al., 2003) Supplemental Table 2. Fatty Acid Composition of Total Wild-Type using a randomized design. A similar planting of wild-type and DsRed and cbr1-2 Leaf and Root Tissues. plants was used as a control. Plants were tied individually prior to silique yellowing, and DsRed plants were removed before harvesting to avoid Supplemental Table 3. Complementation of Homozygous cbr1-2 possible contamination. The number of DsRed seeds per plant was Restores 18:3 Levels in Mature T2 Seeds. 3064 The Plant Cell

Supplemental Table 4. Complementation of the cbr1-3 and cbr1-4 Chen, M., and Thelen, J.J. (2013). ACYL-LIPID DESATURASE2 is Mutants. required for chilling and freezing tolerance in Arabidopsis.PlantCell 25: 1430–1444. Clough, S.J., and Bent, A.F. (1998). Floral dip: A simplified method for ACKNOWLEDGMENTS Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16: 735–743. We thank Michael Knoblauch and staff of the Franceschi Microscopy Curtis, M.D., and Grossniklaus, U. (2003). A Gateway cloning vector and Imaging Center at Washington State University for advice and set for high-throughput functional analysis of genes in planta. Plant assistance with microscopy techniques. We also thank Deirdre Fahy for Physiol. 133: 462–469. her revisions of the figures. This work has been funded by the USDA- Dailey, H.A., and Strittmatter, P. (1980). Characterization of the National Institute for Food and Agriculture Competitive Grants Program interaction of amphipathic cytochrome b5 with stearyl coenzyme A (Grant 2010-65115-20393) and by the Agricultural Research Center at desaturase and NADPH:cytochrome P-450 reductase. J. Biol. Washington State University. L.L.W. was partially supported by NIH, Chem. 255: 5184–5189. National Institute for General Medical Sciences through institutional de Vetten, N., ter Horst, J., van Schaik, H.P., de Boer, A., Mol, J., training Grant T32-GM008336. and Koes, R. (1999). A cytochrome b5 is required for full activity of flavonoid 39,59-hydroxylase, a cytochrome P450 involved in the formation of blue flower colors. Proc. Natl. Acad. Sci. USA 96: 778–783. AUTHOR CONTRIBUTIONS Duncan, O., Taylor, N.L., Carrie,C.,Eubel,H.,Kubiszewski- L.L.W., J.G.W., J.E.M., and J.B. designed the research. L.L.W., J.E.M., Jakubiak, S., Zhang, B., Narsai, R., Millar, A.H., and Whelan, J. and R.K. performed the experiments and analyzed the data. L.L.W., J.G.W., (2011). Multiple lines of evidence localize signaling, morphology, and J.B. wrote the article. and lipid biosynthesis machinery to the mitochondrial outer membrane of Arabidopsis. Plant Physiol. 157: 1093–1113. Dunkley, T.P., et al. (2006). Mapping the Arabidopsis organelle Received May 1, 2013; revised July 29, 2013; accepted August 7, 2013; proteome. Proc. Natl. Acad. Sci. USA 103: 6518–6523. published August 30, 2013. Dürr, U.H.N., Waskell, L., and Ramamoorthy, A. (2007). The cytochromes P450 and b5 and their reductases—Promising targets for structural studies by advanced solid-state NMR spectroscopy. Biochim. Biophys. Acta 1768: 3235–3259. REFERENCES Eastmond, P.J. (2006). SUGAR-DEPENDENT1 encodes a patatin domain triacylglycerol lipase that initiates storage oil breakdown in Alexander, M.P. (1969). Differential staining of aborted and germinating Arabidopsis seeds. Plant Cell 18: 665–675. nonaborted pollen. Stain Technol. 44: 117–122. Enoch, H.G., and Strittmatter, P. (1979). Cytochrome b5 reduction Alonso, J.M., et al. (2003). Genome-wide insertional mutagenesis of by NADPH-cytochrome P-450 reductase. J. Biol. Chem. 254: Arabidopsis thaliana. Science 301: 653–657. – Bando, S., Takano, T., Yubisui, T., Shirabe, K., Takeshita, M., and 8976 8981. Finn, R.D., McLaughlin, L.A., Hughes, C., Song, C.L., Henderson, Nakagawa, A. (2004). Structure of human erythrocyte NADH-cytochrome b5 reductase. Acta Crystallogr. D. Biol. Crystallogr. 60: 1929–1934. C.J., and Roland Wolf, C. (2011). Cytochrome b5 null mouse: Berkowitz, O., Jost, R., Pollmann, S., and Masle, J. (2008). a new model for studying inherited skin disorders and the role of Characterization of TCTP, the translationally controlled tumor unsaturated fatty acids in normal homeostasis. Transgenic Res. 20: – protein, from Arabidopsis thaliana. Plant Cell 20: 3430–3447. 491 502. Bewley, M.C., Marohnic, C.C., and Barber, M.J. (2001). The structure Fukuchi-Mizutani, M., Mizutani, M., Tanaka, Y., Kusumi, T., and and biochemistry of NADH-dependent cytochrome b5 reductase are Ohta, D. (1999). Microsomal electron transfer in higher plants: now consistent. Biochemistry 40: 13574–13582. Cloning and heterologous expression of NADH-cytochrome b5 – Boavida, L.C., and McCormick, S. (2007). Temperature as reductase from Arabidopsis. Plant Physiol. 119: 353 362. a determinant factor for increased and reproducible in vitro pollen Fulda,M.,Schnurr,J.,Abbadi,A.,Heinz,E.,andBrowse,J.(2004). germination in Arabidopsis thaliana.PlantJ.52: 570–582. Peroxisomal Acyl-CoA synthetase activity is essential for seedling Bogs, J., Ebadi, A., McDavid, D., and Robinson, S.P. (2006). development in Arabidopsis thaliana. Plant Cell 16: 394–405. Identification of the flavonoid hydroxylases from grapevine and their Gleave, A.P. (1992). A versatile binary vector system with a T-DNA regulation during fruit development. Plant Physiol. 140: 279–291. organisational structure conducive to efficient integration of cloned Boutté,Y.,andGrebe,M.(2009). Cellular processes relying on sterol DNA into the plant genome. Plant Mol. Biol. 20: 1203–1207. function in plants. Curr. Opin. Plant Biol. 12: 705–713. Gostincar, C., Turk, M., and Gunde-Cimerman, N. (2010). The Browse, J., McConn, M., and James, D., Jr.,., and Miquel, M. evolution of fatty acid desaturases and cytochrome b5 in eukaryotes. (1993). Mutants of Arabidopsis deficient in the synthesis of J. Membr. Biol. 233: 63–72. alpha-linolenate. Biochemical and genetic characterization of the Guengerich, F.P. (2005). Reduction of cytochrome b5 by NADPH- endoplasmic reticulum linoleoyl desaturase. J. Biol. Chem. 268: cytochrome P450 reductase. Arch. Biochem. Biophys. 440: 204–211. 16345–16351. Hayashi, M., Toriyama, K., Kondo, M., and Nishimura, M. (1998). Chen,M.,Markham,J.E.,andCahoon,E.B.(2012). Sphingolipid 2,4-Dichlorophenoxybutyric acid-resistant mutants of Arabidopsis D8 unsaturation is important for glucosylceramide biosynthesis have defects in glyoxysomal fatty acid beta-oxidation. Plant Cell 10: and low-temperature performance in Arabidopsis.PlantJ.69: 183–195. 769–781. Heazlewood, J.L., Tonti-Filippini, J.S., Gout, A.M., Day, D.A., Chen, M., Markham, J.E., Dietrich, C.R., Jaworski, J.G., and Whelan, J., and Millar, A.H. (2004). Experimental analysis of the Cahoon, E.B. (2008). Sphingolipid long-chain base hydroxylation is Arabidopsis mitochondrial proteome highlights signaling and regulatory important for growth and regulation of sphingolipid content and components, provides assessment of targeting prediction programs, and composition in Arabidopsis.PlantCell20: 1862–1878. indicates plant-specific mitochondrial proteins. Plant Cell 16: 241–256. CBR1 and Male Gametophyte Function 3065

Hultquist, D.E., Xu, F., Quandt, K.S., Shlafer, M., Mack, C.P., Till, Muralla, R., Lloyd, J., and Meinke, D. (2011). Molecular foundations G.O., Seekamp, A., Betz, A.L., and Ennis, S.R. (1993). Evidence of reproductive lethality in Arabidopsis thaliana.PLoSONE6: that NADPH-dependent methemoglobin reductase and administered e28398. riboflavin protect tissues from oxidative injury. Am. J. Hematol. 42: Nagano,M.,Ihara-Ohori,Y.,Imai,H.,Inada,N.,Fujimoto,M., 13–18. Tsutsumi, N., Uchimiya, H., and Kawai-Yamada, M. (2009). Hwang, Y.T., Pelitire, S.M., Henderson, M.P., Andrews, D.W., Dyer, Functional association of cell death suppressor, Arabidopsis Bax

J.M., and Mullen, R.T. (2004). Novel targeting signals mediate the inhibitor-1, with fatty acid 2-hydroxylation through cytochrome b5. sorting of different isoforms of the tail-anchored membrane protein Plant J. 58: 122–134. cytochrome b5 to either endoplasmic reticulum or mitochondria. Nagano, M., Takahara, K., Fujimoto, M., Tsutsumi, N., Uchimiya, Plant Cell 16: 3002–3019. H.,andKawai-Yamada,M.(2012). Arabidopsis sphingolipid fatty Jiang,L.,Yang,S.L.,Xie,L.F.,Puah,C.S.,Zhang,X.Q.,Yang,W.C., acid 2-hydroxylases (AtFAH1 and AtFAH2) are functionally differentiated Sundaresan, V., and Ye, D. (2005). VANGUARD1 encodes a pectin in fatty acid 2-hydroxylation and stress responses. Plant Physiol. 159: methylesterase that enhances pollen tube growth in the Arabidopsis 1138–1148. style and transmitting tract. Plant Cell 17: 584–596. Okuley, J., Lightner, J., Feldmann, K., Yadav, N., Lark, E., and Kearns,E.V.,Hugly,S.,andSomerville,C.R.(1991). The role of Browse, J. (1994). Arabidopsis FAD2 gene encodes the enzyme cytochrome b5 in delta 12 desaturation of oleic acid by microsomes that is essential for polyunsaturated lipid synthesis. Plant Cell 6: of safflower (Carthamus tinctorius L.). Arch. Biochem. Biophys. 284: 147–158. 431–436. Palanivelu, R., and Tsukamoto, T. (2012). Pathfinding in angiosperm König, S., Feussner, K., Schwarz, M., Kaever, A., Iven, T., reproduction: Pollen tube guidancebypistilsensuressuccessful Landesfeind, M., Ternes, P., Karlovsky, P., Lipka, V., and double fertilization. Wiley Interdiscip. Rev. Dev. Biol. 1: 96–113. Feussner, I. (2012). Arabidopsis mutants of sphingolipid fatty acid Percy, M.J., and Lappin, T.R. (2008). Recessive congenital a-hydroxylases accumulate ceramides and salicylates. New Phytol. methaemoglobinaemia: cytochrome b(5) reductase deficiency. Br. 196: 1086–1097. J. Haematol. 141: 298–308. Kumar,R.,Wallis,J.G.,Skidmore,C.,andBrowse,J.(2006). A Percy, M.J., McFerran, N.V., and Lappin, T.R. (2005). Disorders of mutation in Arabidopsis cytochrome b5 reductase identified by oxidised haemoglobin. Blood Rev. 19: 61–68. high-throughput screening differentially affects hydroxylation and Rosso, M.G., Li, Y., Strizhov, N., Reiss, B., Dekker, K., and desaturation. Plant J. 48: 920–932. Weisshaar, B. (2003). An Arabidopsis thaliana T-DNA mutagenized Li, Y., Beisson, F., Pollard, M., and Ohlrogge, J. (2006). Oil content population (GABI-Kat) for flanking sequence tag-based reverse of Arabidopsis seeds: The influence of seed anatomy, light and genetics. Plant Mol. Biol. 53: 247–259. plant-to-plant variation. Phytochemistry 67: 904–915. Routaboul, J.M., Benning, C., Bechtold, N., Caboche, M., and Li-Beisson, Y., et al. (2013). Acyl-lipid metabolism. The Arabidopsis Lepiniec, L. (1999). The TAG1 locus of Arabidopsis encodes for a Book 11: e0161, doi/10.1199/tab.0161. diacylglycerol acyltransferase. Plant Physiol. Biochem. 37: 831–840. Liu, C.M., and Meinke, D.W. (1998). The titan mutants of Arabidopsis Sessions, A., et al. (2002). A high-throughput Arabidopsis reverse are disrupted in mitosis and cell cycle control during seed genetics system. Plant Cell. 14: 2985–2994. development. Plant J. 16: 21–31. Shanklin, J., and Cahoon, E.B. (1998). Desaturation and related Markham, J.E., and Jaworski, J.G. (2007). Rapid measurement of modifications of fatty acids. Annu. Rev. Plant Physiol. Plant Mol. sphingolipids from Arabidopsis thaliana by reversed-phase high- Biol. 49: 611–641. performance liquid chromatography coupled to electrospray ionization Shockey, J.M., Dhanoa, P.K., Dupuy, T., Chapitala, D.C., Mullen, R. tandem mass spectrometry. Rapid Commun. Mass Spectrom. 21: T.,andDyer,J.M.(2005). Cloning, functional analysis, and 1304–1314. subcellular localization of two isoforms of NADH: Cytochrome b(5) Markham, J.E., Lynch, D.V., Napier, J.A., Dunn, T.M., and Cahoon, reductase from developing seeds of tung (Vernicia fordii). Plant Sci. E.B. (2013). Plant sphingolipids: Function follows form. Curr. Opin. 169: 375–385. Plant Biol. 16: 350–357. Slack, C.R., Roughan, P.G., and Terpstra, J. (1976). Some properties of McConn, M., and Browse, J. (1998). Polyunsaturated membranes are a microsomal oleate desaturase from leaves. Biochem. J. 155: 71–80. required for photosynthetic competence in a mutant of Arabidopsis. Smith,M.A.,Cross,A.R.,Jones,O.T.,Griffiths, W.T., Stymne, S., Plant J. 15: 521–530. and Stobart, K. (1990). Electron-transport components of the Michaelson, L.V., Zäuner, S., Markham, J.E., Haslam, R.P., 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase Desikan, R., Mugford, S., Albrecht, S., Warnecke, D., Sperling, (delta 12-desaturase) in microsomal preparations from developing P., Heinz, E., and Napier, J.A. (2009). Functional characterization safflower (Carthamus tinctorius L.) cotyledons. Biochem. J. 272: of a higher plant sphingolipid D4-desaturase: Defining the role of 23–29. sphingosine and sphingosine-1-phosphate in Arabidopsis. Plant Smith, M.A., Dauk, M., Ramadan, H., Yang, H., Seamons, L.E., Physiol. 149: 487–498. Haslam, R.P., Beaudoin, F., Ramirez-Erosa, I., and Forseille, L. Miquel, M., and Browse, J. (1992). Arabidopsis mutants deficient (2013). Involvement of Arabidopsis ACYL-COENZYME A DESATURASE- in polyunsaturated fatty acid synthesis. Biochemical and genetic LIKE2 (At2g31360) in the biosynthesis of the very-long-chain characterization of a plant oleoyl-phosphatidylcholine desaturase. monounsaturated fatty acid components of membrane lipids. Plant Physiol. J. Biol. Chem. 267: 1502–1509. 161: 81–96. Mizutani, M., and Ohta, D. (1998). Two isoforms of NADPH: Sousa, E., Kost, B., and Malhó, R. (2008). Arabidopsis cytochrome P450 reductase in Arabidopsis thaliana. Gene structure, phosphatidylinositol-4-monophosphate 5-kinase 4 regulates pollen heterologous expression in insect cells, and differential regulation. Plant tube growth and polarity by modulating membrane recycling. Plant Physiol. 116: 357–367. Cell 20: 3050–3064. Mori,T.,Kuroiwa,H.,Higashiyama,T.,andKuroiwa,T.(2006). Sperling, P., Zähringer, U., and Heinz, E. (1998). A sphingolipid GENERATIVE CELL SPECIFIC 1 is essential for angiosperm fertilization. desaturase from higher plants. Identification of a new cytochrome Nat. Cell Biol. 8: 64–71. b5 fusion protein. J. Biol. Chem. 273: 28590–28596. 3066 The Plant Cell

Stuitje, A.R., Verbree, E.C., van der Linden, K.H., Mietkiewska, E.M., Welti,R.,Li,W.,Li,M.,Sang,Y.,Biesiada,H.,Zhou,H.E., Nap, J.P., and Kneppers, T.J. (2003). Seed-expressed fluorescent Rajashekar, C.B., Williams, T.D., and Wang, X. (2002). Profiling proteins as versatile tools for easy (co)transformation and high-throughput membrane lipids in plant stress responses. Role of phospholipase D functional genomics in Arabidopsis. Plant Biotechnol. J. 1: 301–309. alpha in freezing-induced lipid changes in Arabidopsis.J.Biol. Tiedje, C., Holland, D.G., Just, U., and Höfken, T. (2007). Proteins Chem. 277: 31994–32002. involved in sterol synthesis interact with Ste20 and regulate cell Woody, S.T., Austin-Phillips, S., Amasino, R.M., and Krysan, P.J. polarity.J.CellSci.120: 3613–3624. (2007). The WiscDsLox T-DNA collection: an arabidopsis community Wallis, J.G., Watts, J.L., and Browse, J. (2002). Polyunsaturated fatty acid resource generated by using an improved high-throughput T-DNA synthesis: What will they think of next? Trends Biochem. Sci. 9: 467–473. sequencing pipeline. J. Plant Res. 120: 157–165. Wang,Y.,Zhang,W.Z.,Song,L.F.,Zou,J.J.,Su,Z.,andWu,W.H. Zhou,J.J.,Liang,Y.,Niu,Q.K.,Chen,L.Q.,Zhang,X.Q.,andYe,D. (2008). Transcriptome analyses show changes in (2013). The Arabidopsis general transcription factor TFIIB1 (AtT- to accompany pollen germination and tube growth in Arabidopsis. FIIB1) is required for pollen tube growth and endosperm de- Plant Physiol. 148: 1201–1211. velopment. J. Exp. Bot. 64: 2205–2218. Supplemental Data. Wayne et al. Plant Cell. (2013). 10.1105/tpc.113.113324

Supplemental Data for: Cytochrome b5 Reductase Encoded by CBR1 is Essential for a Functional Male Gametophyte in Arabidopsis Laura L. Wayne, James G. Wallis, Rajesh Kumar, Jonathan E. Markham and John Browse Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340.

Supplemental Figure 1. Further RT-PCR Analysis of Homozygous cbr1-2.

(A) Nested RT-PCR of cbr1-2 leaf and inflorescence (Inflores.) tissues. Secondary PCR (2) used 20-fold dilution of primary RT-PCR (1) products. Control amplification of ACT8 demonstrates that the RNA was intact. (B) RT-PCR of cbr1-2 using CBR1 P6 primer (see Figure 1) and a series of primers designed to the left border of the T-DNA insertion (primers described in Supplemental Table 4, online), amplifying up to 1300bp within the T-DNA. Right lane, molecular weight markers. In the left lane, no template was added to the reaction.

1 Supplemental Data. Wayne et al. Plant Cell. (2013). 10.1105/tpc.113.113324

60% 60% A Phosphatidylcholine B Phosphatidyl- 50% 50% ethanolamine 40% 40%

30% 30% mol % mol % 20% 20%

10% 10%

0% 0% 16:0 16:1 18:0 18:1 18:2 18:3 16:0 16:1 18:0 18:1 18:2 18:3

60% C Phosphatidylinositol 50% 40%

30%

mol % 20% 10%

0% 16:0 16:1 18:0 18:1 18:2 18:3

Supplemental Figure 2. Fatty Acid Compositions of Major Seed Phospholipids.

Individual phospholipids were separated from lipid extracts of mature seed of wild type (white bars) and cbr1-2 (black bars). The amount of each phospholipid (g/g fresh weight +/- SD) for wild type (WT) and cbr1-2 are listed below.

(A) Phosphatidylcholine, WT, 1084+/-192; cbr1-2, 998+/-5 (B) Phosphatidylethanolamine, WT, 403+/-30; cbr1-2, 382+/-58 (C) Phosphatidylinositol, WT, 381+/-35; cbr1-2, 316+/-43

In Supplementary Figures 2 - 4 the fatty acid are designated X:Y where X is the number of carbon atoms in the acyl chain and Y the number of C=C double bonds. In Supplemental Figure 4 the sphingolipid longchain bases are designated: d18:0, dihydroxy 18-carbon chain with no desaturation (sphinganine)

t18:0, trihydroxy 18-carbon chain with no desaturation (phytosphingosine)

d18:1 and t18:1, indicate desaturation at the  or 8 position in the carbon chain

(Markham, et al., 2013)

2 Supplemental Data. Wayne et al. Plant Cell. (2013). 10.1105/tpc.113.113324

40% A Phosphatidylcholine 70% D Monogalactosyldiacylglycerol 35% 60% 30% 50% 25% % %

40% 20%

mol mol 30% 15% 10% 20% 5% 10% 0% 0% 16:0 16:1 16:2 16:3 18:0 18:1 18:2 18:3 16:0 16:1 16:2 16:3 18:0 18:1 18:2 18:3

40% B Phosphatidylethanolamine 70% E Digalactosyldiacylglycerol 35% 60% 30% 50% 25% % % 40% 20%

mol 30% mol 15% 20% 10% 10% 5% 0% 0% 16:0 16:1 16:2 16:3 18:0 18:1 18:2 18:3 16:0 16:1 16:2 16:3 18:0 18:1 18:2 18:3

40% C Phosphatidylglycerol 35% 30% 25% % 20% mol 15% 10% 5% 0% 16:0 16:1 16:2 16:3 18:0 18:1 18:2 18:3

Supplemental Figure 3. Fatty Acid Compositions of Major Leaf Glycerolipids.

Individual lipids were separated from extracts of rosette leaf tissue from wild-type (white bars) and

cbr1-2 (black bars). The amount of each phospholipid (g/g fresh weight +/- SD) for wild type (WT) and cbr1-2 are listed below.

(A) Phosphatidylcholine, WT, 492+/-12; cbr1-2, 523+/-95 (B) Phosphatidylathanolamine, WT, 265+/-13; cbr1-2, 277+/-59 (C) Phosphatidylglycerol, WT, 328+/-33; cbr1-2 , 333+/-46 (D) Monogalactosyldiacylglycerol, WT, 1463+/-157; cbr1-2, 706+/-48 (E) Digalactosyldiacylglycerol, WT, 679+/-13; cbr1-2, 998+/-5

(In phosphatidylglycerol 16:1 is the 3, trans isomer.)

3 Supplemental Data. Wayne et al. Plant Cell. (2013). 10.1105/tpc.113.113324

100% A Sphingolipid LCB 50% B Sphingolipid Fatty acids 80% 40%

60% 30%

40% 20%

20% 10%

0% 0% d18:0 d18:1 t18:0 t18:1 16:0 18:0 20:0 20:1 22:0 22:1 24:0 24:1 26:0 26:1

100% C GIPC LCB 50% D GIPC Fatty acid

80% 40%

60% 30%

40% 20%

20% 10%

0% 0% d18:0 d18:1 t18:0 t18:1 16:0 18:0 20:0 20:1 22:0 22:1 24:0 24:1 26:0 26:1

E Glucosylceramide LCB F Glucosylceramide Fatty acid 100% 50%

80% 40%

60% 30%

40% 20%

20% 10%

0% 0% d18:0 d18:1 t18:0 t18:1 16:0 18:0 20:0 20:1 22:0 22:1 24:0 24:1 26:0 26:1

Supplemental Figure 4. Compositional Analyses of the Longchain Base (LCB) and Fatty

Acid Components of Sphingolipids from Leaves of Wild Type (white bars) and cbr1-2 (black bars). The amount of each sphingolipid (nmole/g dry weight +/- SD) for wild type (WT) and cbr1-2 are listed below.

(A,B) Total sphingolipids (C,D) Glycosyl inositolphosphoceramide (GIPC) , WT, 384+/-109; cbr1-2, 437+/-130 (E,F) Glucosylceramide, WT, 175+/-6; cbr1-2, 169+/-48 (G,H) Ceramide, WT, 66+/-6; cbr1-2, 115+/-10 (I,J) Hydroxyceramide, WT, 56+/-8; cbr1-2, 69+/-14

The fatty acids shown include α-hydroxy derivatives, which predominate in glucosylceramides and GIPCs.

4 Supplemental Data. Wayne et al. Plant Cell. (2013). 10.1105/tpc.113.113324

100% G Ceramide LCB 50% H Ceramide Fatty acid

80% 40%

60% 30%

40% 20%

20% 10%

0% 0% d18:0 d18:1 t18:0 t18:1 16:0 18:0 20:0 20:1 22:0 22:1 24:0 24:1 26:0 26:1

100% I Hydroxyceramide LCB 50% J Hydroxyceramide Fatty acid

80% 40%

60% 30%

40% 20%

20% 10%

0% 0% d18:0 d18:1 t18:0 t18:1 16:0 18:0 20:0 20:1 22:0 22:1 24:0 24:1 26:0 26:1

5 Supplemental Data. Wayne et al. Plant Cell. (2013). 10.1105/tpc.113.113324

Supplemental Table 1. List of Primers.

Name Primer P1 GATACCGAGTTTCTCCGAACC P2 ACTTGGAACATGGGAGTGATG P3 CACTCCTGCCAGTTGCTAGA P4 CACCACCATCCCATACTTCC P5 ATGGATACCGAGTTTCTCCGA P6 TCAGAACTGGAATTGCATCTCC P7 GGAGACCATCTTGCCGTAAA 5'PRM AGCCATCTAATGGACGCCTA LB3 TAGCATCTGAATTTCATAACCAATCTCGATACAC WiscLB AACGTCCGCAATGTGTTATTAAGTTGTC GKLB ATAATAACGCTGCGGACATCTACATTTT ACT8 FW ATGGCCGATGCTGATGAC ACT8 RV TTAGAAGCATTTTCTGTGGACAATG LB4 CGTTTATTTCGGCGTGTAGG LB5 GTTGACGATGGTGCAGACC LB6 CTTCAGCAGGTGGGTGTAGAG LB7 GCTCAACACATGAGCGAAAC CBR1-PRM* ATAAGAATGCGGCCGCCCACTTATCGAACCAAACCTTG CBR1-3UTR* TATCAAATCGCCGGCGACGTATAGACTAGTAGCCAAAAATTGATAG CBR1-PRM-topo CACCCTTAAGCCACTTATCGAACCAAACCTTG CBR1-PRM-end GGCTTCTCTTAACCCAAATGAA

* Not1 restriction site nucleotides are underlined.

6 Supplemental Data. Wayne et al. Plant Cell. (2013). 10.1105/tpc.113.113324

Supplemental Table 2. Fatty Acid Composition of Total WT and cbr1-2 Leaf and Root Tissues. Leaf Root Fatty Acids WT cbr1-2 WT cbr1-2 16:0 15.0±0.2 15.2±.02 25.7±0.7 22.8±0.6 16:1 3.2±0.0 2.7±0.2 n/da n/d 16:2 0.5±0.0 0.5±0.1 n/d n/d 16:3 12.8±0.1 13.2±0.1 n/d n/d 18:0 1.5±0.0 1.4±0.0 5.3±0.8 3.4±0.5 18:1 1.7±0.0 1.5±0.2 5.6±0.3 11.4±1.2 18:2 12.8±0.2 12.4±1.0 42.1±1.0 42.3±0.6 18:3 52.5±0.4 53.1±1.4 21.4±0.2 20.2±0.3 Fatty acids were derivatized directly from leaf and root tissues. Data are means +/- SE for three biological replicates an/d, not detected, <0.5%.

Supplemental Table 3. Complementation of Homozygous cbr1-2 Restores 18:3 Levels in Mature T2 Seeds. Fatty Acid (relative %) Line 16:0 16:1 18:0 18:1 18:2 18:3 20:0 20:1 20:2 22:1 cbr1-2 +CBR1 #7 8.4 0.4 2.7 18.0 30.9 17.7 1.8 17.3 1.5 1.3 cbr1-2 +CBR1 #12 8.4 0.4 2.8 14.6 29.6 21.2 2.0 17.9 1.8 1.4 cbr1-2 9.0 0.6 3.8 28.8 30.5 5.7 2.1 17.7 0.9 0.9 cbr1-2 10.1 0.7 3.5 29.3 30.8 7.3 2.0 15.7 0.7 0.0 WT 8.2 0.4 2.7 14.7 30.3 20.7 1.9 17.8 1.8 1.5 WT 8.6 0.4 2.8 16.0 30.1 20.2 1.9 17.3 1.6 1.3 Homozygous cbr1-2 was transformed with the CBR1 genomic clone (+CBR1) and compared with the parental cbr1-2 and WT. Two representative lines (from 16 total) are shown.

7 Supplemental Data. Wayne et al. Plant Cell. (2013). 10.1105/tpc.113.113324

Supplemental Table 4. Complementation of The cbr1-3 and cbr1-4 Mutants. Progeny Mutant Line cbr1/cbr1 Total CBR1/cbr1-3 3-3 +CBR1a 5 27 -CBR1 0 11 CBR1/cbr1-3 3-4 +CBR1 6 55 -CBR1 0 15 CBR1/cbr1-4 45-1 +CBR1 8 35 -CBR1 0 7 Heterozygous plants were transformed with the CBR1 genomic clone and the progeny genotyped by PCR. a+CBR1 indicates progeny containing the genomic transgene.

8