Photosynthetic Performance and Fertility Are ... - Plant Physiology

Photosynthetic Performance and Fertility Are Repressed in GmAOX2b Antisense Soybean1[OA] Tsun-Thai Chai, Daina Simmonds, David A. Day, Timothy D. Colmer, and Patrick M. Finnegan* School of Plant Biology and Institute of Agriculture, Faculty of Natural and Agricultural Sciences, University of Western Australia, Crawley, Western Australia 6009, Australia (T.-T.C., T.D.C., P.M.F.); Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada K1A 0C6 (D.S.); and Australian Research Council Centre of Excellence in Plant Energy Biology, School of Biological Sciences, Flinders University, Adelaide, South Australia 5001, Australia (D.A.D.)

The alternative oxidase (AOX) is a cyanide-resistant oxidase that provides an alternative outlet for electrons from the respiratory electron transport chain embedded in the inner membrane of plant mitochondria. Examination of soybean (Glycine max) plants carrying a GmAOX2b antisense gene showed AOX to have a central role in reproductive development and fecundity. In three independently transformed antisense lines, seed set was reduced by 16% to 43%, whereas ovule abortion increased by 1.2- to 1.7-fold when compared with nontransgenic transformation control plants. Reduced fecundity was associated with reductions in whole leaf cyanide-resistant, salicylhydroxamic acid-sensitive respiration and net photosynthesis, but there was no change in total respiration in the dark. The frequency of potential fertilization events was reduced by at least one-third in the antisense plants as a likely consequence of prefertilization defects. Pistils of the antisense plants contained a higher proportion of immature-sized, nonfertile embryo sacs compared with nontransgenic control plants. Increased rates of pollen abortion in vivo and reduced rates of pollen germination in vitro suggested that the antisense gene compromised pollen development and function. Reciprocal crosses between antisense and nontransgenic plants revealed that pollen produced by antisense plants was less active in fertilization. Taken together, the results presented here indicate that AOX expression has an important role in determining normal gametophyte development and function.

The electron transport system embedded in the plant inner mitochondrial membrane contains two terminal oxidases, cytochrome oxidase and alternative oxidase (AOX). AOX is a cyanide-resistant enzyme that provides an alternative exit point for electrons entering the respiratory pathway. Electrons that exit the ubiquinol pool through AOX bypass the protontranslocating respiratory complexes III and IV, contributing less to the formation of the electrochemical proton gradient across the inner mitochondrial membrane than electrons passing along the cytochrome pathway. Consequently, ATP production relative to oxygen consumption is lower for the alternative pathway than for the cytochrome pathway (Finnegan et al., 2004). 1

This work was supported by the Australian Research Council (grants to P.M.F. and D.A.D.), by an International Postgraduate Research Scholarship from the Australian Government, Department of Education, Employment, and Workplace Relations, and by a University Postgraduate Award from the University of Western Australia (to T.-T.C.). * Corresponding author; e-mail [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Patrick M. Finnegan ([email protected]). [OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.149294 1638

A major role for AOX in plants appears to be in the avoidance of oxidative stress by maintaining a low level of reduction of the ubiquinone pool of the respiratory chain (Maxwell et al., 1999), and AOX gene expression is induced strongly by most stress conditions (Clifton et al., 2005). Variable engagement of AOX may also contribute to maintaining a constant cellular energy charge, allowing plants to maintain stable growth rates under changing environmental conditions (Hansen et al., 2002; Moore et al., 2002). AOX is likely to allow continued turnover of the tricarboxylic acid (TCA) cycle when the activity of the cytochrome pathway is restricted by high cellular energy charge or low ADP availability (Lambers, 1985; Wagner and Krab, 1995). Thus, AOX may help plant organs adjust to developmentally programmed changes in energy demand and biosynthetic requirements (Lennon et al., 1995; McCabe et al., 1998; Millar et al., 1998). AOX may help optimize photosynthesis by dissipating excess reductant generated in chloroplasts, protecting the photosynthetic apparatus against photoinhibition (Yoshida et al., 2006). AOX-mediated heat generation is crucial to the pollination biology of thermogenic species. Thermogenesis was proposed to facilitate the evaporation of scent compounds to attract pollinators (Meeuse and Raskin, 1988) and/or to provide an optimum temperature for floral development (Seymour and SchultzeMotel, 1998). Recently, thermogenesis was proposed to prevent low-temperature damage to developing

Plant PhysiologyÒ, March 2010, Vol. 152, pp. 1638–1649, www.plantphysiol.org Ó 2010 American Society of Plant Biologists

Alternative Oxidase, Plant Fertility, and Photosynthesis

pollen (Onda et al., 2008) and to optimize pollen germination and pollen tube growth in skunk cabbage (Symplocarpus renifolius), ensuring successful fertilization in early spring (Seymour et al., 2009). The role of AOX in reproduction in nonthermogenic plants is unclear. Tissue- and stage-specific production of AOX in the anthers of Phaseolus vulgaris suggests that AOX is important to plant reproduction, specifically during microsporogenesis and microgametogenesis (Johns et al., 1993). Strong production of AOX in the tapetum and developing microspores of a fertile petunia (Petunia hybrida) line, but not in a cytoplasmic male-sterile line (Conley and Hanson, 1994), and increased pollen abortion in AOX antisense (AS) tobacco (Nicotiana tabacum; Kitashiba et al., 1999) suggest an involvement of AOX in pollen development. The uncoupling by AOX of carbon flux through the TCA cycle from adenylate control (Palmer, 1976; Day et al., 1995) may be important in maintaining biosynthesis in the tapetum and sporogenous tissues during pollen formation (Conley and Hanson, 1995). Recently, AtAOX1a and AtAOX1b transcripts have been detected in Arabidopsis (Arabidopsis thaliana) pollen following germination and tube elongation (Wang et al., 2008), suggesting that AOX may have a role in pollen tube growth. The female gametophyte, or embryo sac, is crucial to most phases of plant sexual reproduction, from pollen tube guidance to initiating embryo and seed development (Yadegari and Drews, 2004). The expression of a GUS reporter gene driven by the GmAOX1 promoter in the entire carpel and by the GmAOX2b promoter in the stigma of Arabidopsis suggests that AOX may also have a role in the development or function of the female reproductive tissues (Thirkettle-Watts et al., 2003). A recent comparison of the transcript profiles from wild-type and mutant pistils lacking an embryo sac revealed that AtAOX1a was an embryo sacexpressed gene (Johnston et al., 2007), providing further circumstantial evidence that AOX may be involved in embryo sac development and/or function. In plants, AOX is encoded by a small gene family whose members can be divided into two groups, AOX1 and AOX2, based on amino acid sequence identity (Considine et al., 2002). Some species have undergone a proliferation of AOX1-type genes, while others have not. Arabidopsis, for example, has four AOX1-type genes and one AOX2-type gene, while soybean (Glycine max) only has one AOX1-type gene and two AOX2-type genes, GmAOX2a and GmAOX2b (Whelan et al., 1996; Finnegan et al., 1997; Considine et al., 2002). So far, no biochemical differences have been observed for the isoforms encoded by the two gene types. However, it is generally an AOX1-type gene that is stress induced in those plants where the induced isoform of AOX has been identified. In soybean, the expression of GmAOX2a and GmAOX2b proteins is differentially regulated in a developmental and tissue-dependent manner. GmAOX2a is found predominantly in green cotyledons and leaves, while Plant Physiol. Vol. 152, 2010

GmAOX2b occurs in roots, cotyledons, and leaves (Finnegan et al., 1997; McCabe et al., 1998; Djajanegara et al., 2002). To give us the best opportunity for gaining insights into the role of AOX on a whole plant basis, we AS suppressed the expression of GmAOX2b, the most widely distributed and abundant form of AOX in soybean. It was found that AOX is an important determinant of plant fecundity at the level of both the male and female gametophytes.

RESULTS Leaf AOX Protein Expression Was Repressed in GmAOX2b AS Soybean

Three independent soybean transgenic events were recovered after the introduction of a full-length GmAOX2b cDNA in the AS orientation under the control of the 35S promoter. The lines derived from the primary transformants were named 3B10, 6C2, and 6C5. To recover homozygous individuals, each primary transformant was propagated by self-pollination. For line 3B10, a homozygous individual was identified in the T3 generation by PCR (data not shown). For lines 6C2 and 6C5, persistent non-Mendelian transgene segregation occurred to the T6 generation (data not shown) and homozygous AS plants were not recovered. The plants used as controls in the following experiments were nontransgenic segregants isolated from each transformed line. The lack of the AS transgene was confirmed by PCR. The AS plants were somewhat shorter and had lower green leaf area relative to the nontransgenic controls, but flower number and flowering time were unchanged. A detailed growth analysis of the plants will be presented elsewhere (T.-T. Chai, T.D. Colmer, and P.M. Finnegan, unpublished data). The effect of the transgene on AOX protein abundance in green leaves was determined for the AS transformed lines. In each of the 66 T3 generation AS plants examined, immunoreactive proteins were detected at 34 and 36 kD (Fig. 1), corresponding to the soybean GmAOX2a and GmAOX2b proteins, respectively (Finnegan et al., 1997). The product of the stressinducible GmAOX1 gene (Tanudji et al., 1999) was not detected in these plants. No plant was found to completely lack GmAOX2b or GmAOX2a protein in the leaves. Porin, an outer mitochondrial membrane protein, was also detected and used as a loading control for total mitochondrial protein (Ribas-Carbo et al., 2005). Representative immunoblots for four AS plants and four control plants are shown (Fig. 1). The GmAOX2b-to-porin signal ratios were used to compare the relative abundance of GmAOX2b between nontransgenic and AS plants (Fig. 2A). Within each line, the individual nontransgenic control plant giving the greatest signal from the 36-kD GmAOX2b protein was benchmarked to 100%. The GmAOX2b signal from all the other plants within the line was 1639

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Figure 1. Immunoblot detection of GmAOX proteins and porin in crude membrane preparations from youngest fully expanded leaves of nontransgenic (N) and GmAOX2b AS (AS) siblings of line 6C5. Immunodetection and densitometry were carried out as described in “Materials and Methods.” Crude membrane preparations were immunoblotted in duplicate and probed with either an anti-AOX or an antiporin antibody. The sizes of the proteins detected are shown on the left. The numbers below the panels are the signal ratios of total GmAOX2 (GmAOX2b + GmAOX2a) to porin, calculated from the intensities of the respective bands.

compared with that benchmark. Using this approach, the abundance of GmAOX2b protein relative to porin in the leaves of nontransgenic soybean varied over a 1.5-fold range. The mean value for the three sets of control plants was 77% to 91% of the benchmark value for the relevant line (Fig. 2A). In the AS plants, the mean abundance of GmAOX2b was 53% to 70% of the benchmark value for the line. The mean values for control and AS plants were not significantly different based on the Mann-Whitney U test. However, the variation among the AS plants was much greater than in the controls. In each line, a subset of AS plants had a GmAOX2b abundance that fell within the range of the control plants, indicating that there was no AS suppression in these individuals. In contrast, the abundance of GmAOX2b in some AS plants was nearly 65% lower than the mean for the relevant nontransgenic controls. In lines 3B10 and 6C5, about half of the AS plants had less GmAOX2b than all the control plants (Fig. 2A), while in line 6C2, the decrease in GmAOX2b was more severe, with 60% of AS plants having less of this protein than all the control plants. These results, taken in light of the known variability in AS suppression of gene expression (Penarrubia et al., 1992; Zhang et al., 1997), suggest that the GmAOX2b AS gene suppressed GmAOX2b expression in a highly variable manner. The abundance of GmAOX2a among the nontransgenic controls plants ranged from 67% to 79% of the benchmark, while the mean value for the AS plants ranged from 62% to 73% of the benchmark (Fig. 2B). While the difference in the mean abundance of GmAOX2a protein between control and AS plants was not significant, 25% of AS plants from lines 3B10 and 6C5 and 50% of AS plants from line 6C2 had less GmAOX2a protein than all the control plants. This result suggested that cosuppression of the GmAOX2a gene by the GmAOX2b AS gene was occurring in at least some individual plants. Overall, the total abundance of GmAOX2 protein relative to porin in the 1640

leaves of nontransgenic soybean varied over a 2-fold range, with a mean value that was 78% to 80% of the benchmark (Fig. 2C). In the AS plants, the mean abundance of total GmAOX2 was 59% to 70% of the benchmark. Line 6C2 had the greatest proportion of individual plants showing suppression of total GmAOX2 protein, as 60% of AS plants had less GmAOX2 than all of the control plants. However, the most severely suppressed plants belonged to line 3B10 (Fig. 2C). The impact of the AOX repression on respiration was determined. Surprisingly, despite the fact that AOX is a mitochondrial respiratory protein, no significant differences were found in the leaf dark respira-

Figure 2. Relative abundance of GmAOX2b (A), GmAOX2a (B), and GmAOX2b + GmAOX2a (C) in GmAOX2b AS soybean. The relative amounts of AOX2 protein and porin in the youngest fully expanded leaves from nontransgenic (N) and GmAOX2b AS (AS) plants were determined as described in “Materials and Methods.” In the boxwhisker plots, the boxes represent plants between the 25th and 75th percentiles, with the whiskers extending to the data extremes but excluding outliers (circles). Outliers are defined within the R statistical software package as data points lying a distance of more than 1.5 times the interquartile range below the 25th or above the 75th percentile. The interquartile range is the difference between the 25th and 75th percentiles. The continuous and broken lines within each box indicate the median and mean values, respectively. The numbers of plants analyzed are shown in parentheses. Plant Physiol. Vol. 152, 2010


Figure 3. Oxygen consumption rates of the leaf tissues of nontransgenic (N) and GmAOX2b AS (AS) plants from line 3B10. A, Total respiration rates. B, KCN-resistant, SHAM-sensitive respiration rates. C, KCN-resistant, SHAM-sensitive respiration rates expressed as a percentage of total respiration. Total respiration rates were determined by subtracting residual oxygen uptake rates (measured in the presence of 2.5 mM KCN and 10 mM SHAM) from initial oxygen uptake rates (measured in the absence of inhibitors). KCN-resistant, SHAM-sensitive respiration rates were determined by subtracting residual oxygen uptake rates from oxygen uptake rates measured in the presence of 2.5 mM KCN. All measurements were carried out using leaf strips cut from the youngest, fully expanded leaves from N and AS plants as described in “Materials and Methods.” The box-whisker plots were as described in the legend to Figure 2, with the numbers of plants analyzed shown in parentheses. The asterisk indicates the AS group where the mean value was significantly different from the nontransgenic group as determined by the Mann-Whitney U test. DW, Dry weight.

tion rates between AS and nontransgenic control plants from all three lines using gas exchange (data not shown). Total leaf respiration was also similar in control and AS plants from homozygous line 3B10 when measured using an oxygen electrode (Fig. 3A). When KCN was added to the oxygen electrode assays, there was a stimulation of oxygen uptake in the control leaf tissues but not in the AS leaf tissues (Fig. 3B). When the KCN-resistant salicylhydroxamic acid (SHAM)-sensitive respiration rate was expressed as a percentage of total respiration rate, alternative pathway activity was found to be significantly lower in AS than in control leaf tissues (Fig. 3C). Plant Productivity Was Repressed in GmAOX2b AS Soybean

The physiological impact of AOX repression in soybean was assessed using gas exchange to deterPlant Physiol. Vol. 152, 2010

mine net photosynthesis. Net photosynthetic rates were reduced up to 32% in leaves attached to the main stem and up to 43% in leaves attached to branches of AS plants from lines 3B10 and 6C2 (Fig. 4). In line 6C5, net photosynthetic rates were suppressed only in the branch leaves. In making these measurements, it was noted that stomatal conductance (gs) had a strong, positive correlation (r2 = 0.81, P , 0.05) with net photosynthetic rate across the three lines. More importantly, however, the internal CO2 concentration (Ci) did not differ between AS and nontransgenic control plants across the three lines but was a constant 293 6 2 mmol mol21 (data not shown). The constant value for Ci indicated that the observed variations in gs did not affect the rate of substrate delivery to photosynthesis. The repression in photosynthetic activity was also accompanied by a corresponding reduction in the concentration of photosynthetic pigments (data not shown). Seed yield was determined for AS and control plants in the T5 generation. The nontransgenic control plants had similar seed yields across the three lines. The average number of seeds produced by the AS plants of all three lines was reduced compared with the nontransgenic control plants (Fig. 5). Homozygous AS plants from line 3B10 were most severely affected, having an average 43% reduction in seed yield compared with controls, with all plants producing less seed than any of the control plants. Heterozygous AS plants from lines 6C2 and 6C5 had a lower depression in seed yield. Line 6C2 was least affected, with only 25% of plants producing less seed than any of the control plants. There was a greater variation in seed yield in line 6C2 compared with line 6C5, correlating with the greater variation in relative AOX protein amount in the leaves of line 6C2 (Fig. 2). The undeveloped ovules produced by the same plants used in the seed yield analysis were counted

Figure 4. Net CO2 assimilation rates in GmAOX2b AS soybean. The net CO2 uptake rates of youngest fully expanded leaves on the main stem (black bars) and on the branches (white bars) of the nontransgenic (N) and GmAOX2b AS (AS) plants were determined as described in “Materials and Methods.” All plants within a line were assessed on the same day, while each line was assessed on a different day. Data are means 6 SE (n = 10 plants). Asterisks indicate mean values that were significantly different (P , 0.05) according to Student’s t test from the corresponding value determined for nontransgenic control plants. 1641

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Figure 5. Seed production in GmAOX2b AS soybean. The seeds produced by nontransgenic (N) and GmAOX2b AS (AS) plants from the three transgenic lines grown as described in “Materials and Methods” were counted. The numbers in parentheses indicate the number of plants. The box-whisker plots were as described in the legend to Figure 2. Asterisks indicate AS groups where the mean values were significantly different from the nontransgenic groups as determined by the Mann-Whitney U test.

to determine whether the reduced seed set in the GmAOX2b AS plants was due to increased frequency of ovule abortion. More than 1,800 pods were examined for each transgenic line, and no attempt was made to determine the fertilization status of the aborted ovules. Across the three lines, the proportion of aborted ovules was increased by 1.2- to 1.7-fold in the AS plants compared with nontransgenic controls (Fig. 6). Although greater variation was seen again among the 6C2 AS plants, the proportion of ovule abortion in lines 6C2 and 6C5 was generally similar and intermediate between that of line 3B10 and the nontransgenic controls.

revealed 165 ovules that were penetrated by a pollen tube. Each penetrated ovule was entered by only one pollen tube, and the pollen-ovule interaction appeared normal. The ovules that were entered by a tube were randomly positioned within the pistil, indicating that the reduced frequency of fertilization in the AS plants was not due to restricted pollen tube elongation in vivo. Confirmation that cross-pollination had not occurred during the hand-pollination process, and that flowers had been selected before anther dehiscence, came from the examination of pistils from 30 emasculated but nonpollinated flowers (10 from each line). All lacked both pollen grains on the stigma and pollen tube growth within the ovary. When the pistils of the hand-pollinated flowers were examined, two types of ovules were observed: those containing elongated, mature-sized embryo sacs (Fig. 8A) and those containing shorter, immature-sized embryo sacs (Fig. 8B). The ovules containing immature-sized embryo sacs were 10% to 20% smaller than ovules with mature-sized embryo sacs and were randomly positioned in the pistils. The small ovules were unlikely to provide productive fertilization events, as none of the 138 small ovules observed was penetrated by a pollen tube, even though pollen tubes were often lying adjacent to the ovule. The flowers of AS plants from lines 3B10 and 6C5 contained 30% to 40% fewer mature-sized ovules than the nontransgenic controls (Fig. 8C). There was no difference in the number of mature-sized ovules in line 6C2, which may explain to some degree the variation seen in the phenotypes between this line and the other heterozygous line 6C5. It is unlikely that the immature ovules came from immature flowers. The presence of at least some

Plant Fertility Was Compromised in GmAOX2b AS Soybean

Plants from the three AS lines were self-pollinated by hand to determine whether the reduced seed set in the AS plants was associated with a reduction in fertilization events or a disruption in a postfertilization process. After germination, pollen tubes within the pistil were visualized by staining for callose, a major carbohydrate constituent of pollen tube walls (Li et al., 1999). Potential fertilization events were scored when a pollen tube was observed that had penetrated an ovule and come into contact with the embryo sac (Fig. 7A). The frequency of potential fertilization events in the flowers of T6 generation AS plants from all three lines was reduced by at least one-third compared with the nontransgenic controls (Fig. 7B). In total, visual examination of 414 ovules from 143 hand-pollinated pistils 1642

Figure 6. Ovule abortions in GmAOX2b AS soybean. The nontransgenic (N) and GmAOX2b AS (AS) soybean plants were the same as those described in the legend to Figure 5. The box-whisker plots were as described in the legend to Figure 2. Asterisks indicate AS groups where the mean values were significantly different from the nontransgenic groups as determined by the Mann-Whitney U test. Plant Physiol. Vol. 152, 2010


three lines that germinated in vitro was reduced by 43% to 50%, compared with the nontransgenic controls. The decrease in pollen germination rate was accompanied by an increase in the proportion of ungerminated pollen grains that were either filled or aborted. The pollen abortion rates across the AS plants from the three lines was 1.6- to 2.1-fold higher than in the respective nontransgenic controls. Again, the severity of the AS phenotype was lowest in line 6C2,

Figure 7. Potential fertilization events in GmAOX2b AS soybean. A, Potential fertilization events, where a pollen tube (PT) had penetrated an ovule (O) through a micropyle (M) and come into contact with the embryo sac (ES), were visualized by staining with decolorized aniline blue. B, Frequency of potential fertilization events expressed as the proportion of ovules examined that were penetrated by a pollen tube as in A. Randomly selected flowers from five nontransgenic (N) and five GmAOX2b AS (AS) plants from each line were hand pollinated with self pollen and analyzed as described in “Materials and Methods.” The numbers in parentheses indicate numbers of ovules examined/number of ovaries examined. Asterisks indicate AS groups where the mean values were significantly different from the nontransgenic groups as determined by the Mann-Whitney U test.

ovules that had attracted pollen tubes indicated that the flowers were mature and receptive to pollen tube penetration (Fig. 7B). Additionally, the ovules within a normal soybean flower reach maturity simultaneously (Kennell and Horner, 1985). The ability of AS pollen to germinate in vitro was tested to determine whether a defect in the male gametophyte also contributed to the reduced seed set in GmAOX2b AS soybean. The pollen sample tested for germination in vitro was subsequently stained with Alexander’s stain (Alexander, 1969) to determine the likely viability status of the grains that did not germinate. The pollen germination rate for the nontransgenic controls was similar across the three lines, as was the proportion of ungerminated pollen grains that were either filled or aborted (Fig. 9). The proportion of pollen grains from T6 generation AS plants from the Plant Physiol. Vol. 152, 2010

Figure 8. Availability of ovules containing mature-sized embryo sacs in GmAOX2b AS soybean. A and B, Micrographs of mature-sized (A) and immature-sized (B) embryo sacs in soybean ovules. Mature, fertile embryo sacs were elongated and in contact with the internal opening of the micropyle. Bars = 100 mm. C, Percentage of ovules from handpollinated nontransgenic (N) and GmAOX2b AS (AS) flowers that contained mature-sized embryo sacs. The numbers of ovaries and ovules examined are given in Figure 7B. 1643

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50% of the mature ovules examined, regardless of whether the receptive female was from an AS or nontransgenic plant. Mature AS ovules were just as receptive to pollen as their nontransgenic counterparts. The effectiveness of the hand-pollination procedure in making these crosses was confirmed by the absence of pollen and pollen tubes from 20 emasculated flowers that were not pollinated.

DISCUSSION Figure 9. Pollen viability in GmAOX2b AS soybean. The proportions of pollen grains that germinated (black bars), that were filled but did not germinate (white bars), or that were aborted (gray bars) were determined for nontransgenic (N) and GmAOX2b AS (AS) plants in the three GmAOX2b AS lines. At least 2,500 pollen grains sampled from 10 randomly chosen, fully opened flowers per plant were cultured in vitro and stained as described in “Materials and Methods.” Data are means 6 SE (n = 5 plants). Asterisks indicate AS groups that were significantly different (P , 0.05) from the corresponding nontransgenic group as determined by Student’s t test.

The defective pollen and ovule phenotypes in GmAOX2b AS soybean demonstrated that AOX expression is important to gametophytic development and function. Reduction in the frequency of fertilization events in the AS plants indicated that potential seed set was already compromised before the fertilization stage. Notably, development and function of both male and female gametophytes were defective in the AS plants. Consequently, the reduced fertilization efficiency, and by extension the reduced fecundity, in the GmAOX2b AS soybean can be attributed to prefertilization defects. Perturbation of mitochondrial function in plants often leads to problems in male fertility, illustrated by the numerous examples where mutations to the mitochondrial genome cause cytoplasmic male sterility and the associated abnormalities in the male organs and pollen (Linke and Borner, 2005). However, our observations of defects in both male and female gametophytes in GmAOX2b AS soybean are in agreement with emerging evidence that disruptions in mitochondrial function can impair the formation and development of female, as well as male, gametophytes. In Arabidopsis, disruption of the SDH1-1 gene, which encodes a subunit of mitochondrial succinate dehydrogenase (respiratory complex II), caused aberrant mitosis in the microspores but also arrested the fusion of polar nuclei during embryo sac development (Leon et al., 2007). Mutation of the Arabidopsis RPL21M gene, which encodes the L21 protein of the mitochondrial 50S ribosome, not only increased pollen abortion but also prevented normal nuclear fusion

which had the lowest proportion of aborted pollen and the highest proportion of filled, ungerminated pollen of the AS lines. The interactions between GmAOX2b AS and control gametes were tested in vivo in reciprocal crosses using flowers from T6 generation plants. For ease of interpretation, only the homozygous AS line 3B10 was used, as every gamete produced will carry a GmAOX2b AS allele. After hand pollination, pollen tubes that penetrated flower ovules were visualized by aniline blue staining to determine the frequency of potential fertilization events. Data were expressed as the proportion of mature ovules that were penetrated for two reasons: the nontransgenic and AS plants of line 3B10 differed in the abundance of mature ovules produced (Fig. 8), and only mature ovules were likely to be fertilized. Nontransgenic pollen was equally successful in penetrating mature ovules from both AS and nontransgenic plants, with about 70% of mature ovules being penetrated in each case (Table I). In contrast, AS pollen was able to penetrate less than

Table I. Pollen-ovule interactions in reciprocal crosses of nontransgenic and GmAOX2b AS plants Hand pollination was carried out on line 3B10 AS and control plants as described in “Materials and Methods.” For each cross, flowers were randomly chosen from five nontransgenic and five AS plants. Pollen Donor

Female Parent

No. of Hand-Pollinated Pistils

Total Ovules

Mature Ovulesa

Nontransgenic Nontransgenic AS AS

Nontransgenic AS Nontransgenic AS

31 36 31 29

96 110 93 85

85 53 66 50

Proportion of Mature Ovules Penetrated by a Pollen Tubeb % mature ovules per pistil

a

Ovules containing mature-sized embryo sacs. pistils). 1644

b

Data are means 6

70 66 42 49 SE

6 6 6 6

5.5 7.8 6.8 7.7

(n = no. of hand-pollinated

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during embryo sac development and during double fertilization (Portereiko et al., 2006). The increase in defective male gametophyte development in AOX AS soybean was observed as an increase in pollen abortion. An increase in the proportion of aborted pollen was also reported for an AOX AS mutant of tobacco (Kitashiba et al., 1999). In tobacco, the aborted pollen grains appeared shrunken and shriveled and lacked starch grains. In GmAOX2b AS soybean, the aborted pollen largely lacked cytoplasm. There was also a higher proportion of unaborted and apparently fully filled pollen that was functionally compromised, as there was a clear reduction in the proportion of this class of pollen that germinated in vitro, as well as a reduction in the ability of this pollen to fertilize in vivo. AOX repression in rice (Oryza sativa; K. Toriyama, personal communication) and Arabidopsis (Umbach et al., 2005) did not cause fertility phenotypes. While AOX may have a different role in these species, it is important to note that all previous studies targeted an AOX1-type isoform for suppression. Therefore, it is possible that another AOX homolog than the one tested is involved in gametophyte development in rice and Arabidopsis. In Arabidopsis, this may be the AOX2-type homolog, while in rice it would necessarily be another AOX1type homolog, as rice does not seem to possess an AOX2-type homolog. The compromised pollen phenotype in the GmAOX2b AS soybean was consistent with the proposed role for AOX in microsporogenesis and microgametogenesis (Johns et al., 1993). The Arabidopsis sdh1-1 mutant produced developmentally and functionally defective pollen that was unable to transmit the mutated allele to the next generation (Leon et al., 2007). Non-Mendelian inheritance may be a common phenotype for respiratory mutations that affect both male and/or female gamete production, as it was also observed in lines 6C2 and 6C5. The AS allele in line 3B10 did not follow this non-Mendelian pattern of inheritance but could be recovered in the homozygous state. It may be that the transgene effect elicited by a single AS locus within line 3B10 was not severe enough to trigger the cascade of events that lead to defective pollen. The defect in pollen production seen in the Atsdh1-1 insertional mutant was suggested to be due to a compromise in energy production and the disruption of carbon skeleton supply to biosynthesis in the developing gametophytes (Leon et al., 2007). The same rationale cannot fully explain the defects we observed in the GmAOX2b AS soybean. It seems unlikely that down-regulation of AOX would lead to a decrease in cellular energy charge, as AOX is not involved in energy conservation. Instead, a decrease in AOX activity is likely to allow the energy charge of the cell to increase, increasing the likelihood that harmful reactive oxygen species (ROS) will be produced. An increase in ROS production may have a deleterious effect on pollen formation and may underlie the Plant Physiol. Vol. 152, 2010

phenotype of AOX AS soybean. An increase in the cellular energy charge due to a decrease in AOX activity might lead to a restriction of the TCA cycle, a situation that AOX is thought to ameliorate (Palmer, 1976). The resulting decrease in the supply of carbon skeletons to biosynthesis could potentially occur in both AOX AS soybean and sdh1-1 Arabidopsis and may be particularly important in tissues with high rates of biosynthesis, such as the tapetum and sporogenous tissues (Conley and Hanson, 1995). The possibility that these AOX AS and sdh1-1 mutations exert their phenotypes through the common mechanism of altering TCA cycle activity deserves further attention. The involvement of AOX in female gametophyte development was indicated by the increased frequency of immature-sized embryo sacs in the GmAOX2b AS plants of lines 3B10 and 6C5. The increased abundance of apparently nonfertile embryo sacs suggested that the number of potential fertilization events was already preset at a lower level in the GmAOX2b AS plants, regardless of the level of pollen viability. The presence of immature-sized embryo sacs within smaller ovules may be another common outcome of events that disrupt mitochondrial function. The phenotype was similar to the early disruption in ovule growth seen in Arabidopsis with a mutation in the HUELLENLOS gene, which encodes the L14 protein of the mitochondrial ribosome (Skinner et al., 2001). The Atsdh1-1 mutant of Arabidopsis, with decreased mitochondrial respiratory complex II, is also defective in embryo sac development (Leon et al., 2007). As in pollen formation, AOX may have a role in determining female gametophyte fertility, specifically embryo sac development, by allowing TCA cycle turnover during times of high cellular energy status and/or in limiting ROS formation (Finnegan et al., 2004). AOX may also protect the developing embryo sac from inappropriate triggering of programmed cell death (PCD). AS tobacco cells lacking AOX were more susceptible to cell death-inducing compounds, such as hydrogen peroxide and Cys, than wild-type cells (Robson and Vanlerberghe, 2002; Vanlerberghe et al., 2002). Furthermore, overexpression of AOX limits the progression of the hypersensitive response, a form of PCD, in tobacco leaves (Ordog et al., 2002). During embryo sac development, there are four PCD events in an ovule. The first event begins after the meiotic divisions and ensures that only one megaspore is retained. The second PCD event causes the degeneration of the antipodal cells in the developing embryo sac. The final two PCD events occur prior to fertilization. One synergid cell undergoes PCD, allowing the male gametes to be received, while concurrently PCD directs the degeneration of the surrounding ovular tissues (nucellus) to accommodate the expanding embryo sac (Christensen et al., 1997; Wu and Cheung, 2000). During these PCD events, AOX may protect the cells of a developing embryo sac from oxidative stress or cell death signals produced in the cellular 1645

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neighborhood. It seems likely that limiting oxidative damage is an important aspect of embryo sac development, given that high levels of ascorbate peroxidase were detected in rice egg cells (Uchiumi et al., 2007). There is currently much debate about the nature of the signal that attracts a growing pollen tube into the micropyle (Higashiyama and Hamamura, 2008). It was of interest that none of the small ovules observed in AOX AS soybean was penetrated by a pollen tube. An intriguing possibility is that AOX activity may have a role in generating that signal. Dark respiration rates were unaltered in the leaves of the AS plants, whether measured using gas exchange or an oxygen electrode. This observation is similar to that seen in the leaves of AOX-repressed Arabidopsis (Umbach et al., 2005) and in a cell line of AOX-repressed tobacco (Vanlerberghe et al., 1994). In this study, lower KCN-resistant respiration rates in the AS leaf tissue relative to nontransgenic leaf tissue indicates a lower potential for AOX respiration in the AS plants. This result is consistent with the lower levels of AOX protein abundance in the AS plants. Interestingly, the addition of KCN to oxygen electrode assays stimulated O2 uptake in nontransgenic leaf tissues, an observation previously reported in AOX-overexpressing Arabidopsis plants (Umbach et al., 2005) and in an AOX-overexpressing tobacco cell line (Vanlerberghe et al., 1994). One explanation for this may be that KCN-stimulated O2 uptake occurs in intact tissues due to reduced ATP synthesis following inhibition of the cytochrome respiration (Atkin et al., 2002). The reduction in ATP synthesis would lead to the decline in cellular ATP-to-ADP ratios, which would in turn stimulate glycolysis in a bid to increase the supply of respiratory substrates to the mitochondria. Consequently, the potential for respiratory electron flow would increase. However, increased electron flow could only occur if the AOX capacity in the tissue is high, as is the case in soybean and overexpressing lines, and if O2 uptake by the tissue is substrate limited (Atkin et al., 2002). Photosynthetic performance was repressed in the AOX-repressed soybean. While there was variation in gs between nontransgenic and AS plants, the Ci was the same for all the leaf samples examined (data not shown). Therefore, the reduction in gs in the AS plants was probably of secondary importance in reducing the net CO2 assimilation rates (for review, see Farquhar and Sharkey, 1982) and may represent to some extent a response to the reduced photosynthetic rate (Wong et al., 1979). Thus, we conclude that the reduction in photosynthetic capacity in the AS plants is primarily associated with biochemical or cellular limitations. Such a limitation appears to have arisen from the repression of AOX protein expression. It is unclear whether AOX repression has a direct effect on photosynthesis or an indirect effect, perhaps through the reduced concentration of photosynthetic pigments. Inhibition of AOX also led to reduced photosynthetic rates in leaves of broad bean (Vicia faba) and resulted in 1646

the overreduction of the photosynthetic electron transport chain even under low-light conditions (Yoshida et al., 2006). The reduction in photosynthetic performance may indicate a decrease in assimilate supply during reproductive growth of the AS plants. Thus, it is possible that the observed reduction in seed set in the AS plants was also influenced by decreased assimilate availability during reproductive development. Soybean is known to adjust its reproductive sink size in response to variations in photosynthetic rates that affect source strength or assimilate supply (Heitholt et al., 1986; Vega et al., 2001; Egli, 2005). However, the observed increase in ovule abortion and decrease in pollen viability argue against the possibility that all of the reproductive phenotypes demonstrated by the AS plants were a consequence of repressed photosynthetic performance. Soybean often responds to conditions that alter photosynthetic performance, source and sink relations, or assimilate supply during flowering or the entire reproductive phase by regulating flower abortion or pod set, not the number of seeds per pod and abortion of ovules in developing pods. Drought stress, chilling, manganese deficiency, defoliation, and shading all compromised pod and seed set in soybean (Heenan and Campbell, 1980; Musser et al., 1986; Frederick et al., 1991; Board et al., 1995; Liu et al., 2003; Egli and Bruening, 2005). However, unlike the GmAOX2b AS gene, these conditions do not change the number of seeds per pod, suggesting that the rate of ovule abortion also remains unchanged. Light and CO2 enrichment, conditions that increased soybean pod and seed set, also do not change the number of seeds per pod (Mathew et al., 2000; Ziska et al., 2001). Furthermore, a hand-pollination experiment also demonstrated that in water-stressed soybean, decreased photosynthetic performance was not associated with impaired pollen fertility (Kokubun et al., 2001). GmAOX2 protein was readily detectable in leaves of wild-type soybean grown under normal conditions. In contrast, AOX protein was barely or not detectable in leaves of wild-type Arabidopsis, rice, or tobacco grown under similar conditions (Vanlerberghe et al., 1994; Abe and Toriyama, 2003; Umbach et al., 2005). Therefore, in the latter studies, it was necessary to analyze the effects of AOX AS genes in plants exposed to chilling (Vanlerberghe et al., 1994) or KCN treatment (Umbach et al., 2005) or by screening putative AS calli (Abe and Toriyama, 2003). The relatively high abundance of AOX protein in soybean leaf tissue suggests a greater involvement of AOX in the growth and development of soybean compared with those other three species. This may explain the growth and fertility phenotypes observed in AOX-repressed soybean but not in AOX-repressed Arabidopsis (Umbach et al., 2005; Watanabe et al., 2008), rice (K. Toriyama, personal communication), or tobacco (Vanlerberghe et al., 1994). A greater involvement of AOX in soybean growth and development may also explain the inability to identify AS plants with total suppression of Plant Physiol. Vol. 152, 2010


GmAOX2b. Even in AOX AS Arabidopsis (Umbach et al., 2005), tobacco (Vanlerberghe et al., 1994), and rice (Abe and Toriyama, 2003), plants completely lacking AOX were seldom recovered. Importantly, the modest decreases in AOX protein obtained in AOX AS soybean were enough to reduce photosynthetic performance, attenuate plant fecundity, and disrupt gametophyte development and function. In total, these results are consistent with the hypothesis that a threshold amount of AOX protein is essential for plant cell viability (Umbach et al., 2005). In summary, our findings in AOX AS soybean indicate that AOX is important in sustaining plant fertility and normal gametophyte development and function. Interestingly, a role for AOX in plant sexual reproduction was previously only well established in thermogenic species, which produce extremely high amounts of AOX protein in the reproductive tissues. Analysis of the effects of AOX repression in other nonthermogenic species that express AOX protein in much lower, but still readily detectable, amounts under normal growth conditions should help clarify and better define the role of AOX in fertility and gametophytic fitness in plants.

MATERIALS AND METHODS Transgenic Plant Material and Growth Conditions A full-length GmAOX2b cDNA (Finnegan et al., 1997; GmAOX2b was originally named GmAOX3) was excised as a single fragment and ligated downstream of the cauliflower mosaic virus 35S promoter in pCAMBIA1301 (CAMBIA). The AS orientation of the insert relative to the 35S promoter was confirmed by DNA sequencing. The GmAOX2b AS construct was introduced into soybean (Glycine max) via biolistics (PDS-1000/He Particle Delivery System; Bio-Rad Laboratories). The transformation, selection, and plant regeneration protocols were described previously (Simmonds, 2003). Briefly, proliferative embryogenic cultures of soybean cv X5 (X2650-7-2-3; Simmonds and Donaldson, 2000) were cobombarded with GmAOX2b AS and hygromycin resistance gene constructs. Transgenic events were selected and maintained on 55 mg L21 hygromycin. Excised embryos were matured on antibiotic-free medium, air desiccated, and converted on B5 solid medium (Gamborg et al., 1968). Upon transfer to soil, plants designated the T0 generation were grown in a controlled-environment growth chamber. T0 plants carrying the GmAOX2b AS gene were identified by PCR and brought to seed. The plants derived from these seeds (the T1 generation) were the predecessors of plants used in this study. Seed was germinated and plants were grown one per pot in 3 L of commercial potting mix (SSM2500 Potting Mix 2; Richgro Garden Products) at 25°C under natural daylight. Pot positions were randomized. The photoperiod was extended to 16 h with supplementary fluorescent lighting. The plants were watered every other day. Fertilizer (Thrive All Purpose Plant Food; Yates Australia) was applied every 4 weeks. The number of seeds produced per plant and the proportion of aborted ovules (ovules that failed to develop to mature seeds) were recorded.

Identification of GmAOX2b AS Plants Genomic DNA was extracted from fresh leaf discs, and PCR assays were carried out using a commercial kit and the reagents and protocol provided (REDExtract-N-Amp Plant PCR kit; Sigma-Aldrich). The primers used to detect the GmAOX2b AS cDNA were AOX3-F and AOX3-R (Finnegan et al., 1997). The thermal cycling parameters were one cycle of 95°C for 3 min; 40 cycles of 95°C for 30 s, 48°C for 1 min, and 72°C for 1.5 min; and one cycle of 72°C for 10 min. PCR products were separated by electrophoresis on agarose gels.


Immunodetection of AOX Crude mitochondrial membranes were prepared using a modification of the method of Day et al. (1985). Approximately 1 g of leaves from the youngest, fully expanded trifoliolate of plants at the three-trifoliolate stage were harvested and homogenized in 3 volumes of 300 mM Suc, 25 mM sodium pyrophosphate decahydrate, 2 mM disodium EDTA, 1% (w/v) polyvinylpyrrolidone-10, 1% (w/v) bovine serum albumin, and 20 mM sodium ascorbate, pH 7.5. Homogenates were filtered through three Miracloth (Calbiochem) discs fitted into a 10-mL syringe and subjected to differential centrifugation (Day et al., 1985). The isolated mitochondrial membranes were washed with 300 mM Suc, 10 mM TES, and 1 mM Gly, pH 7.5, and resuspended in a small volume of the same buffer, and the protein content was determined (Peterson, 1977). Approximately 25 mg of crude membrane protein was resolved by SDSPAGE and transferred to a nitrocellulose membrane (Protran; Schleicher and Schuell) using a semidry blotting apparatus (Trans-Blot Semi-Dry electrophoretic transfer cell; Bio-Rad; Towbin et al., 1979). Blots were probed with monoclonal antibodies against AOX (AOA; Elthon et al., 1989) or porin. The AOA anti-AOX antibody recognizes a conserved amino acid motif found in all three soybean isoforms (Finnegan et al., 1999). Secondary antibody (ECL AntiMouse IgG, horseradish peroxidase-linked whole antibody from sheep; Amersham Biosciences) was used for chemiluminescence detection using a commercial kit (ECL Western Blotting Detection Reagents; Amersham Biosciences) on films (Hyperfilm; Amersham Biosciences). Signal intensities on the immunoblots were quantified (ImageJ version 1.36 [http://rsb.info.nih.gov/ ij/]; software downloaded on August 7, 2006). Samples from the same set of four nontransgenic control plants were included on each immunoblot to allow direct comparison between experiments.

Measurement of Photosynthesis and Respiration Net photosynthesis and respiration were measured by gas exchange (LI6400 Portable Photosynthesis System; LICOR) on the same fully expanded trifoliolate on the main stem or on a mid-position branch of the plants between 10 AM and 2 PM on bright sunny days 1 week after the initiation of flowering. Measurements were made at a leaf chamber temperature of 25°C, a leaf chamber CO2 concentration of 380 mmol mol21, and a light intensity of 1,500 mmol quanta m22 s21. For the three soybean lines, vapor pressure deficits in the leaf chamber during measurements were 1.83 6 0.01 kPa (line 3B10), 1.67 6 0.01 kPa (line 6C2), and 1.66 6 0.08 kPa (line 6C5). Dark respiration measurements were taken after covering the plants with dark plastic bags overnight at 25°C. Leaf respiration was also measured using an oxygen electrode using leaf strips (approximately 1 mm 3 10 mm) excised from the youngest, fully expanded trifoliolate of main stems between 11 AM and 2 PM 1 week after the initiation of flowering. Excised leaf strips (approximately 15 mg) were incubated in the dark for 10 min in assay buffer (15 mM TES and 0.2 mM CaCl2, pH 7.0; Umbach et al., 2005) before transfer to a 4-mL glass chamber containing fresh air-saturated assay buffer. Oxygen uptake was monitored using an oxygen microelectrode (MicroResp EL; Unisense) in the dark at 25°C as described previously (Colmer and Pedersen, 2008). Assay buffer was supplemented with 2.5 mM KCN to inhibit the cytochrome oxidase pathway and with 10 mM SHAM to inhibit the AOX pathway. Optimal inhibitor concentrations were determined by titration experiments. Leaf samples were recovered from the assay and oven dried at 60°C for 48 h before determining the dry mass.

Pollen Viability Fully open flowers were collected randomly from each plant on the day of anthesis. After the petals were removed, the anthers were dabbed into 30 mL of 15% (w/v) Suc, 0.03% (w/v) Ca(NO3)2·4H2O, and 0.01% (w/v) H3BO3 (Salem et al., 2007) on a glass slide to release the pollen. The slide was incubated at 30°C in darkness for 24 h. A drop of Alexander’s stain (Alexander, 1969) was added, and all the pollen grains were examined with a light microscope. A pollen grain was scored as having germinated if the length of the pollen tube exceeded the diameter of the pollen grain. Pollen grains that did not germinate but stained green or light red with Alexander’s stain were scored as aborted (Alexander, 1969), while those that stained red were scored as filled but ungerminated.

Hand Pollination and in Vivo Pollen Tube Growth Flowers that were expected to open the next day were emasculated (Fehr, 1980) in the evening. The following morning, the emasculated flowers were

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hand pollinated (Fehr, 1980) with pollen from other flowers on the same plant. Emasculated flowers that were not pollinated were used as controls. After 48 h, the hand-pollinated flowers were excised and fixed in ethanol:glacial acetic acid (3:1, v/v) for 24 h at 4°C. The pistils were dissected from the flowers, and the trichomes were removed. The pistils were cleared in 4 N NaOH for 24 h at 20°C, washed in deionized water for 30 min, stained in decolorized aniline blue (0.005% [w/v] aniline blue in 0.05 M sodium phosphate, pH 11) for 24 h, mounted in glycerol (Shivanna and Rangaswamy, 1992), and examined by fluorescence microscopy (Axioplan microscope with Plan-Neofluar Pol objectives, BP-365/12 excitation filter, FT395 beam splitter, LP397 barrier filter; Carl Zeiss Australasia).

Statistical Analyses All statistical analyses were performed using Microsoft Excel 2003. Box plots were prepared with the R software version 2.7.0 (http://www.r-project. org/; software downloaded on April 22, 2008). The Student’s t test was used to analyze data that were normally distributed, as determined by Lilliefors’ test; otherwise, the Mann-Whitney U test was used.

ACKNOWLEDGMENTS We thank Nicolas Taylor for preparing the AOX monoclonal antibody; Sheryl Hubbard for transforming soybean; Mingren Shi and Guijun Yan for statistical advice; Erik Veneklaas for useful discussions on our photosynthesis data; and Ole Pedersen for providing the oxygen electrodes and assistance during the leaf respiration measurements. Received October 12, 2009; accepted January 19, 2010; published January 22, 2010.

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