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CROP BREEDING & GENETICS

Field Evaluation of Transgenic Wheat Expressing a Modified ADP-Glucose Pyrophosphorylase Large Subunit

^a Dep. of Plant Sciences and Plant Pathology, Montana State Univ., Bozeman, MT 59717-3150
^b Dow AgroSciences, 9330 Zionsville Rd., Bldg. 306, C1-249, Indianapolis, IN 46268

^* Corresponding author (mgiroux{at}montana.edu)

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Wheat yield is influenced by the efficacy of seed starch biosynthetic enzymes. ADP-glucose pyrophosphorylase (AGP) catalyzes a rate-limiting step in seed starch biosynthesis. We transformed the hard spring wheat (Triticum aestivum L.) cultivar Hi-Line with a modified maize AGP large subunit sequence (Sh2r6hs) to increase AGP activity. In previously described growth chamber studies, Sh2r6hs conditioned increased AGP activity, seed yield, and plant size. The primary objective of this study was to determine whether a similar yield enhancement could be detected under field conditions. Sh2r6hs transgenics were field tested over four growing years, in three locations, with varying planting density and irrigation. The results indicate that significant yield increases were more likely to occur in space-planted, irrigated environments than densely planted, rainfed environments, suggesting that limited abiotic resources may subsequently limit Sh2r6hs-associated yield enhancement. In elite lines, as in the F₂–derived trials in which tissue culture derived mutations were reduced by out-crossing, Sh2r6hs appears to confer a yield advantage only when field conditions are nonlimiting.

Abbreviations: AGP, adenosine diphosphate (ADP) glucose pyrophosphorylase • BT2, protein expressed by the AGP small subunit gene (Bt2) • SH2 and SH2R6HS, proteins expressed by unaltered and altered maize AGP large subunit genes (Sh2 and Sh2r6hs, respectively) • SKCS, single kernel characterization system

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
THE PRODUCTION and allocation of sugars produced during photosynthesis are controlled by both source and sink tissues. Source tissues generate carbohydrates, which are shipped to sink tissues for growth, development, or storage. A primary sink product in wheat is starch stored in the endosperm, which accounts for approximately 70% of seed weight. Several recent studies have attempted to alter sink strength by targeting specific enzymes involved in starch production. A rate-limiting step in starch biosynthesis is controlled by ADP-glucose pyrophosphorylase (AGP) (Stark et al., 1992; Giroux et al., 1996), which converts Glc-1-P and ATP to ADP-glucose and pyrophosphate (Espada, 1962). Studies in maize (Zea mays L., Giroux et al., 1996), potato (Solanum tuberosum L., Stark et al., 1992), wheat (Smidansky et al., 2002), and rice (Oryza sativa L., Sakulsingharoj et al., 2004; Smidansky et al., 2003) have shown the importance of AGP in controlling sink strength.

AGP is an allosterically regulated enzyme activated by 3-phosphoglyceric acid (3-PGA) and inhibited by orthophosphate (P_i) (Preiss and Romeo, 1994). AGP in plants is a heterotetramer, composed of two large and two small subunits (Morell et al., 1987). Shrunken-2 (Sh2) and Brittle-2 (Bt2) encode the AGP large and small subunit in maize, respectively (Bhave et al., 1990; Bae et al., 1990). Several Sh2 isoalleles were generated by means of the transposable element Dissociation (Ds) (Giroux et al., 1996). One of these isoalleles, Sh2r6, increased seed weight by 18% (Giroux et al., 1996). The Sh2r6 allele has a serine and a tyrosine residue inserted between amino acid positions 495 and 496 of SH2 (Shaw and Hannah, 1992). This insertion confers reduced allosteric inhibition on the AGP holoenzyme but also decreases AGP stability (Giroux et al., 1996). To increase AGP stability, a second SH2 modification (HS33) was incorporated with r6 into SH2. The HS33 alteration enhances binding between large and small subunits when expressed in E. coli (Greene and Hannah, 1998). Sh2hs33 has a tyrosine substituted for histidine at amino acid 333 of the SH2 coding region (Shaw and Hannah, 1992).

Wheat and rice were transformed with a construct consisting of the Sh2r6hs coding sequence (containing both the r6 and the hs33 mutation), Sh2 promoter, and Sh1 intron sequences (Smidansky et al., 2002, 2003). Lines expressing Sh2r6hs had increased yield and plant size relative to non-Sh2r6hs-expressing lines (Smidansky et al., 2002, 2003). However, the level of Sh2-promoter Sh2r6hs expression compared with native AGP large subunit expression in wheat is relatively low, approximately 20% (Meyer et al., 2004). Therefore, we created a construct containing Sh2r6hs flanked by glutenin regulatory sequences. The GluteninDy10 promoter (Blechl and Anderson, 1996) confers Sh2r6hs mRNA levels that are 20 times higher than those seen with the Sh2 promoter (Meyer et al., 2004). The high SH2R6HS levels resulted in AGP activity with decreased allosteric inhibition, increased sensitivity to allosteric activation, and enhanced subunit stability (Meyer et al., 2004). However, the high SH2R6HS levels conferred by the Glu promoter did not confer higher yields than the Sh2 promoter in growth chamber trials (Meyer et al., 2004). In growth chamber trials of both Sh2 and Glu promoter Sh2r6hs events, we observed seed number per plant increases of roughly 15% concomitant with similar increases in whole plant weight (Meyer et al., 2004).

The growing environment is likely to influence the effect of Sh2r6hs on grain yield. In particular, it is important that plants be vigorous enough to set a high number of seeds because the primary source of yield increase in growth chamber trials is increased seed number per plant (Smidansky et al., 2002; Meyer et al., 2004). Seed set may be affected as early as 30 d before anthesis (Fischer, 1985), and solar radiation levels and pollen survivability heavily influence the number of seeds initiated per head (Thorne and Wood, 1986). In addition to environmental effects, the transformation process itself can also negatively affect yield. Transgenic wheat plants created via particle bombardment have been shown to have relatively poor field performance. A population expressing a wheat streak mosaic virus resistance gene yielded significantly lower than the untransformed wild-type variety (Sharp et al., 2002). Similarly, we have shown that 16 of 19 transgenic lines containing seed-specific overexpression of puroindolines have reduced yield relative to an untransformed control (Meyer and Giroux, 2006). Therefore, a yield enhancing transgene would have to increase seed starch synthesis and overcome transformation-associated yield losses. The negative background effect of mutations generated by the transformation process may be mitigated by backcrossing transgenic lines to the original source variety. Transgene locus positive and negative segregants are expected to have a similar number of unlinked mutations but backcrossing would not alleviate any negative impacts of the transgene locus itself.

Our results from growth chamber experiments has suggested that the modified AGP enzyme produced by the Sh2r6hs transgene results in increased yield in wheat. The objective of the experiments reported in this paper was to determine the impact of Sh2r6hs on field-grown lines tested in several environments.

	MATERIALS AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Transformation and Molecular Screening
Three plasmid constructs were used for these experiments. Construct pRQ101A contains the selectable marker gene Bar and confers resistance to the herbicides bialaphos, 4-[hydroxy(methyl)phosphinoyl]-L-homoalanyl- L -alanyl- L -alanine (Meiji Seika Kaisha Ltd., Japan) and glufosinate, 2-amino-4-(hydroxy-methyl-phosphoryl)-butanoic acid (AgrEvo USA Company, Wilmington, DE). CaMV 35S promoter and nopaline synthase (NOS) terminator sequences flank the 5' and 3' ends of Bar, respectively. Construct pSh2r6hs contains a Sh2 promoter, Sh1 intron cassette, Sh2r6hs coding sequence, and NOS terminator (Smidansky et al., 2002). Sh2r6hs is a 1557-bp modified maize Sh2 cDNA with the following two mutations: r6, which encodes an additional tyrosine and serine (Giroux et al., 1996) and hs33, a point mutation at amino acid 333 of tyrosine for histidine (Greene and Hannah, 1998). The r6 and hs33 mutations confer reduced inhibition to phosphate and more stable large subunit-small subunit interactions, respectively. Construct pGS contains Sh2r6hs flanked with Glu Dy10 5' and Glu Dx5 3' regulatory sequences (Meyer et al., 2004; Blechl and Anderson, 1996). Construct pRQ101A was precipitated onto gold particles with either construct pSh2r6hs or pGS and bombarded into Hi-Line (Lanning et al., 1992) callus tissue, using a Biolistic PDS-1000/He Particle Delivery System (BioRad, Hercules, CA). Callus bombardment and tissue-culturing procedures were according to Smidansky et al. (2002). Once transplanted to soil, two-leaf plantlets were tissue-sampled for Sh2r6hs by Southern-blot screening and sprayed with 0.1% glufosinate to identify bar expression.

Identification of Sh2r6hs Positive and Negative Segregants by Progeny Testing of T₁ and F₂-derived Lines
Twenty to 40 Sh2r6hs locus segregating T₁ plants were grown to maturity for each Sh2r6hs T₀ event. Eighteen T₂ plantlets from individual T₁ plants were glufosinate-tested to identify Sh2r6hs homozygous positive and negative T₁ parents. For each event, it was determined that Sh2r6hs cosegregated with Bar as described previously (Smidansky et al., 2002). A T₁ parent was classified as homozygous positive if all of 12 to 18 T₂ seedlings from an individual T₁ plant were herbicide resistant and homozygous negative if all of 12 to 18 T₂ seedlings were herbicide susceptible. Five to 10 Sh2r6hs homozygous positive and negative T₁ plants per transformation event were identified in this manner. Three Glu-promoter T₀ plants (GS 6, GS11, GS12) did not express Bar but were verified Sh2r6hs positive through Southern blotting. T₁ homozygotes from these three plants were selected by western blots using 12 or more T₂ seeds from individual T₁ plants rather than herbicide resistance assays. Segregants showing relatively strong or weak signal on a western blot incubated with an anti-SH2 antibody as described by Meyer et al. (2004) were chosen. Each T₁ homozygote was advanced several generations to produce homozygous seed stocks. Selected Sh2r6hs homozygous positives were then backcrossed to Hi-Line to increase vigor and create F₂–derived Sh2r6hs homozygous positive and negative groups for yield testing. Individual F₂ plants were progeny tested as for T₁s using 12 or more F₃ seedlings from each individual F₂ plant. F₃ plantlets were similarly tested to identify F₂ Sh2r6hs homozygotes. The groups of Sh2r6hs positive and negative homozygotes selected in this manner were ultimately planted in field plots for analysis.

Sh2r6hs Expression Analysis
Immature (21 days post anthesis, 21 dpa) and mature seeds were collected from Sh2r6hs-positive T₁ plants and immature leaf tissue was collected from homozygous Sh2r6hs-positive T₂ plants. The RNA samples were northern-blotted and hybridized with a ³²P-labeled Sh2r6hs probe. Mature seed proteins were western-blotted and incubated with a polyclonal anti-SH2 antibody (Giroux and Hannah, 1994). Leaf tissue DNAs were digested with EcoRI, fractionated on agarose gels, blotted to nylon membranes, and also incubated with a ³²P-labeled Sh2r6hs probe. All three blotting procedures were described previously (Meyer et al., 2004). Table 1 ranks two Sh2-promoter lines and seven Glu-promoter lines according to their Southern, northern, and western blot signal intensities. These intensities represent the levels of Sh2r6hs mRNA and protein present in seed tissue and the relative copy number of Sh2r6hs.

Temuco, Chile 2001–2002
Seven pGS T₀ plants (GS4, GS5, GS6, GS8, GS11, GS12, GS54) were harvested and 24 T₁ seeds per T₀ plant were planted in the greenhouse. Among each group (GS4, GS5, GS6, GS8, GS11, GS12, GS54) of 24 Sh2r6hs-segregating T₁ plants, five Sh2r6hs homozygous positive and five Sh2r6hs homozygous negative plants were identified by the T₂ herbicide testing methods described above. Homozygous T₂ seed (7.5 g) derived from each homozygous T₁ plant was shipped to the Von Baer Farm (Temuco, Chile) and planted in a four-row plot (3-m length, 30-cm row spacing). The elevation at Temuco is 114 m. Before supplementation the soil contained 27 kg ha^–1 of N, 70.8 kg ha^–1 of P, and 40 kg ha^–1 of K. Before planting, 42 kg ha^–1 of N, 70.8 kg ha^–1 of P, and 40 kg ha^–1 of K were applied with an additional 64 kg ha^–1 N applied post germination. Plots were seeded 2 Nov. 2001 and harvested 9 Mar. 2002. Plots received 17.3 cm of rainfall during the growing season and were irrigated two times before and one time shortly after heading with each irrigation event consisting of 5 cm of water. All plots were replicated twice. Each replication was 70 plots, consisting of five positive and five negative homozygous entries for seven GS groups. Yield data were collected on site and plot samples were mailed to Bozeman for protein and single kernel analysis.

Minto, Manitoba 2002
Seven GS events were tested in a randomized complete block design with two replications. Each replication contained seven GS events, and each GS event consisted of six Sh2r6hs positive plots and six Sh2r6hs negative plots. Positive or negative plots were T₃ plants derived from three independent homozygous T₂ parents and were a subset of the lines grown in Chile in 2001–2002. A total of 90 g of homozygous T₃ seed (field grown in Chile in 2001–2002) was planted in nine-row plots (plot dimensions = 5.5 x 1.5 m) by Ag-Quest, Inc. at Minto. The elevation at Minto is 550 m and the soil is a clay loam. Seeding date was 14 May 2002 and harvest date was 13 Sep. 2002. Sixty kilograms per hectare N was applied 6 mo before planting to bring total soil N to 175 kg ha^–1. The trial was irrigated with 5 cm of water 1 wk after heading and received 26.4 cm of rainfall between May and September. Yield data were collected on site and plot samples were mailed to Bozeman for protein and single kernel analysis.

Bozeman 2004, 2005
Two Sh2 promoter events (152–7 and 161–12) and two Glu promoter events (GS5, GS8) were densely planted in irrigated and nonirrigated 3-m rows. All lines were derived from backcrosses of the Sh2r6hs homozygous positive transgenic lines to the original variety Hi-Line. 152–7 and 161–12 F₅ Sh2r6hs homozygous positive and homozygous negative plants were derived from seed pools described in the Bozeman 2001 trial. GS5 and GS8 were F₄ plants derived from T₃ x Hi-Line backcrossing. The F₂ Sh2r6hs homozygotes were selected in the same manner as 161–12, then seed from F₃ plants pooled to generate homozygous F₄ seed for this trial. Seeding density was 12 and 9 g seed per 3 m for irrigated and nonirrigated rows, respectively. Five centimeters of water was applied to irrigated rows 1 wk before and 1 wk after anthesis in 2004, while 7.6 cm of water was applied 1 wk before anthesis in 2005. Rainfall from April to July was 18.7 and 18.0 cm in 2004 and 2005, respectively. No nitrogen was supplemented, and average soil N was 177 kg ha^–1 before planting. Each of the four events was replicated 15 times per environment.

Statistics
Mean values for multiple traits were compared between the positive and negative Sh2r6hs homozygotes described in each of the above experiments. One-tailed, two-sample t tests with equal variances were used to compare means at the 0.05 and 0.01 significance level. Total seed number per hectare was calculated by dividing plot yield by the individual kernel weight average measured for that respective plot. This seed number per plot was then converted to seed number per hectare.

	RESULTS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Select Sh2r6hs events created by means of either the Glu or the Sh2 promoter were chosen for field testing (Table 1). T₁ progeny from four Glu promoter Sh2r6hs T₀ plants (GS4, GS5, GS8, GS54) and two Sh2 promoter Sh2r6hs T₀ plants (152–7, 161–12) segregated for herbicide resistance (data not shown). Three Glu promoter Sh2r6hs T₀ transformants (GS6, GS11, GS12) lack measurable Bar expression, and all T₁ progeny derived from these three events were glufosinate susceptible. Positive and negative Sh2r6hs homozygotes were selected for all lines, either with glufosinate testing or SH2-antibody western blotting using individual seeds, so that field trials could be conducted with plants varying in Sh2r6hs expression. Sh2r6hs homozygous lines from events 152–7 and 161–12 were backcrossed to Hi-Line, and Sh2r6hs positive and negative lines were selected from the crosses. Glu promoter events, on average, have much higher Sh2r6hs mRNA and protein levels than Sh2 promoter events. The increased expression is due to the Glu promoter and not additional copies of Sh2r6hs, since Sh2r6hs Southern blot intensities from Sh2 promoter events were equal or higher to most Glu promoter events. The northern blot signal intensity was comparable to the western blot signal intensity within each event, indicating that Sh2r6hs mRNA levels, not Sh2r6hs copy number, has the strongest impact on SH2R6HS levels.

The 2001 Bozeman trial consisted of Sh2r6hs homozygous positive or homozygous negative F₃ plants for event 161–12 (Table 2) planted under spaced plant density with average final plant density of 10 plants per 3-m row. Because the number of germinating plants differed from row to row, plants were counted in each row and all averages are presented on a single plant basis. Sh2r6hs expression increased yield 12% and biomass 10%. Individual 161–12 plants were plotted according to yield and biomass (Fig. 1 ). There is a strong positive correlation between yield and biomass (r = 0.99). The increase in Sh2r6hs positives 161–12 experimental yield was accompanied by increased shoot weight, therefore, harvest index was unchanged compared with the Sh2r6hs negative control group. Protein content and individual seed weight averages of the Sh2r6hs positive and negative plants were not changed.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Sh2r6hs positive (open circles) vs. Sh2r6hs negative (closed circles) 161–12 yield parameters. Data points represent individual plant seed weights and biomasses obtained from homozygous space-planted F3 plants, Bozeman 2001.

To examine if Sh2r6hs conditioned yield increases would be seen with backcross populations under normal seeding density, we conducted further experiments at Bozeman. Densely seeded experiments containing GS5, GS8, 161–12, and 152–7 were conducted at Bozeman 2004 and 2005. All lines tested in this experiment were derived from backcrosses of the transgenic line to the variety Hi-Line to mitigate background effects of tissue culture and transformation. The data for these two growing seasons were averaged for two irrigated and two rainfed experiments (Table 4); analysis of variance indicted little interaction with environments. Sh2r6hs positive lines from 152–7 flowered significantly later than their negative controls, while Sh2r6hs positive 161–12 flowered significantly earlier than their negative controls. Yield, biomass, and shoot weight was significantly lower for Sh2r6hs positive 161–12 lines relative to negative controls. No significant differences were observed among Sh2r6hs positives and Sh2r6hs negative controls for GS5 and GS8. When Sh2r6hs positive lines from all four groups were compared with Sh2r6hs negative control lines from all four groups, no phenotypic effect was detected.

	DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The primary observation from this study is that significant yield increase occurred only in environments with nonlimiting resources or where overall plant vigor was low because of tissue culture derived mutations. In particular, F₂ backcross derived plants with the Sh2r6hs transgene driven by the Sh2 promoter (161–12) produced more grain than negative controls in a space-planted irrigated experiment at Bozeman. Yield enhancements were also observed in irrigated trials in Manitoba and Chile where the transgenic lines had not been backcrossed to the parent variety. Sh2r6hs did not confer yield enhancement in full density irrigated or rainfed trials in which we used seed derived from backcrosses. Over four trials at Bozeman in 2004 and 2005, Sh2r6hs lines did not condition increased yield. This was true for Sh2r6hs lines driven by both the Sh2 and Glu promoters, respectively. Coupled with previous growth chamber studies that showed a yield advantage to lines with the Sh2r6hs transgene (Smidansky et al., 2002; Meyer et al., 2004), our results suggest that any effect of the transgene may require noncompetitive growth conditions or weak overall plant vigor.

A large number of environmental conditions (including temperature, soil fertility, plant density, and water availability) influence plant growth and metabolism over different developmental stages. When yield enhancement is not detected, it is most likely that uncontrolled conditions are somehow limiting to Sh2r6hs-associated starch biosynthesis. Variables such as mean temperature, soil moisture, and solar radiation may control plant traits such as seed set and ultimately determine whether or not SH2R6HS will increase yield. If seed set is not plentiful, it becomes increasingly difficult to detect a yield enhancement as a result of the modified AGP. Research has indicated that the percentage of initiated florets that develop into mature seeds is as low as 72% (Zhen-Wen et al., 1988). This failure to set seeds is attributed to a deficiency of available assimilates near the time of flowering (Abbate et al., 1998). When we detected significantly higher yields in our trials, these increases were accompanied by increased seed number, suggesting that Sh2r6hs-induced yield increases are dependent on increasing seed number. Average individual kernel size was not increased when seed set was increased. This suggests that the sink strength of individual kernels is maximized, but Sh2r6hs may increase the total quantity of delivered assimilates to more seeds, either to an equal or larger number of heads.

Despite the greater expression levels observed with the Glu-promoter, the results indicate that Glu-promoter events have no phenotypic advantage over Sh2-promoter events. Sh2r6hs GS lines were not more likely to be higher yielding than their controls. Sh2r6hs event 161–12 yield was significantly lower in all 2004–2005 experiments, but this cannot be solely attributed to the Sh2 promoter, since 152–7 yield comparisons were similar to both GS5 and GS8 yield comparisons. The timing of Sh2r6hs expression and field conditions may have a greater impact on Sh2r6hs-induced yield enhancement than the intensity of Sh2r6hs expression alone.

In summary, our results suggest that modification of AGP to decrease allosteric inhibition and increase holoenzyme stability may positively affect grain yield in noncompetitive conditions. A positive impact of the transgene was not observed under less optimal and competitive conditions.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This research was supported by the Montana Agricultural Experiment Station, the Montana Wheat and Barley Committee, the Consortium for Plant Biotechnology Research, Dow AgroSciences, LLC, and USDA Competitive Grant 00–03395.

Received for publication March 10, 2006.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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