Background Developing monocots that accumulate more vegetative tissue protein is one strategy for improving nitrogen-sequestration and nutritive value of forage and silage crops. In soybeans (a dicotyledonous legume), the  vspA  and  B  genes encode subunits of a dimeric vegetative storage protein that plays an important role in nitrogen storage in vegetative tissues. Similar genes are found in monocots; however, they do not accumulate in leaves as storage proteins, and the ability of monocot leaves to support accumulation of an ectopically expressed soybean VSP is in question. To test this, transgenic maize ( Zea Mays  L. Hi-II hybrid) lines were created expressing soybean  vspB  from a maize ubiquitin  Ubi-1  promoter.
Background
Developing monocots that accumulate more vegetative tissue protein is one strategy for improving nitrogen-sequestration and nutritive value of forage and silage crops. In soybeans (a dicotyledonous legume), the  vspA  and  B  genes encode subunits of a dimeric vegetative storage protein that plays an important role in nitrogen storage in vegetative tissues. Similar genes are found in monocots; however, they do not accumulate in leaves as storage proteins, and the ability of monocot leaves to support accumulation of an ectopically expressed soybean VSP is in question. To test this, transgenic maize ( Zea Mays  L. Hi-II hybrid) lines were created expressing soybean  vspB  from a maize ubiquitin  Ubi-1  promoter.vspABZea MaysvspBUbi-1Results From 81 bombardments, 101 plants were regenerated, and plants from five independent lines produced  vspB  transcripts and VSPβ polypeptides. In leaves from seven-week-old plants (prior to flowering), VSPβ accumulated to 0.5% of the soluble leaf protein in primary transgenic plants (R 0 ), but to only 0.03% in R 1  plants. During seed-filling (silage-stage) in R 1  plants, the VSPβ protein was no longer detected in leaves and stems despite continued presence of the  vspB  RNA. The RNA transcripts for this peptide either became less efficiently translated, or the VSPβ protein became unstable during seed-fill.
Results
From 81 bombardments, 101 plants were regenerated, and plants from five independent lines produced  vspB  transcripts and VSPβ polypeptides. In leaves from seven-week-old plants (prior to flowering), VSPβ accumulated to 0.5% of the soluble leaf protein in primary transgenic plants (R 0 ), but to only 0.03% in R 1  plants. During seed-filling (silage-stage) in R 1  plants, the VSPβ protein was no longer detected in leaves and stems despite continued presence of the  vspB  RNA. The RNA transcripts for this peptide either became less efficiently translated, or the VSPβ protein became unstable during seed-fill.vspBvspBConclusion Developmental differences in the accumulation of soybean VSPβ when transgenically expressed in maize show that despite no changes in the  vspB  transcript level, VSPβ protein that is readily detected in leaves of preflowering plants, becomes undetectable as seeds begin to develop.
Conclusion
Developmental differences in the accumulation of soybean VSPβ when transgenically expressed in maize show that despite no changes in the  vspB  transcript level, VSPβ protein that is readily detected in leaves of preflowering plants, becomes undetectable as seeds begin to develop.vspB

Background Although genetic variation for protein content has been found in forage plants, this variability is narrower than that observed for other traits such as digestibility [ 1 ]. Since the major protein components in monocot forage and silage crops are involved in metabolic activity, and hence are not "true" storage proteins, it has been argued that it is not feasible to make major changes in protein quality or protein composition by conventional breeding [ 1 ]. However, genetic engineering may allow improvement in protein quality and content through expression of a storage protein not found in grass vegetative tissue. Genes encoding seed storage proteins of various plant species have been transgenically expressed to test for improvement of nutritional quality. Most experiments were conducted with tobacco and legume species including alfalfa, soybean, canola, clover and lupins. For nuclear-targeted genes, accumulation of these seed storage proteins in vegetative tissue of transgenic plants was either undetectable or very low. These included pea vicilin [ 2 , 3 ], soybean conglycinin [ 4 ], sunflower seed agglutinin [ 5 , 6 ], and phaseolin [ 7 ]. The instability of seed proteins in non-seed tissues of transgenic plants was frequently attributed to protein targeting to protease-rich vacuoles in the vegetative cells, and subsequent degradation [ 5 , 7 , 8 ]. The greatest accumulation of a seed storage protein from a nuclear-targeted gene was achieved using zeins, a maize seed storage protein that is targeted to "protein bodies" directly from the endoplasmic reticulum (ER), thus avoiding the secretory route to the cellular vacuole. Transgenic expression from the CaMV 35S promoter in tobacco resulted in the formation of these protein bodies containing the zein within vegetative tissues [ 8 , 9 ]. Alternatively, "short-circuiting" the protein-targeting route by addition of an ER retention signal to the storage-protein coding region also increased protein accumulation up to 100× [ 5 , 6 , 10 , 11 ]. In many legumes, accumulation of specific vegetative storage proteins (VSPs) in leaves and stems is the main source of increased nitrogen content [ 12 - 14 ]. Use of VSPs instead of seed storage proteins to increase vegetative protein content in monocots may provide an advantage since they have evolved to function in vegetative cell types. Legume vegetative cells that accumulate VSP proteins contain multiple vacuole types and storage proteins are targeted to specific vacuoles where they are not rapidly degraded [ 15 ], and do not interfere with cellular metabolic processes. It is uncertain if monocots can produce similar vacuoles or successfully target a VSP to them. The most studied VSPs are the soybean VSPα and VSPβ proteins, which are lysine-rich glycosylated vacuolar proteins that accumulate abundantly in leaves, stems and pods, but not in seeds [ 12 - 14 ]. Recently, soybean  vspA  and/or  vspB  genes fused to the 35S promoter were expressed in transgenic tobacco to study their accumulation in a heterologous dicot plant. Nuclear targeted genes produced VSP ranging between 2 and 6% of the soluble protein in leaves of the transgenic plants [ 16 ]; whereas, targeting to both the chloroplast and the vacuole within the same plant resulted in VSP comprising greater than 10% soluble protein [ 17 , 18 ]. Soybean VSP is therefore an excellent candidate for use in transgenic improvement of plant protein status, particularly grasses that contain limited levels of lysine [ 16 ]. However, it remains to be determined if VSPs can be expressed and accumulated in monocot plants since storage protein stability is dependent on post-translational events that may differ between monocots and eudicots. This manuscript presents the transgenic expression of soybean VSPβ in the leaves of transgenic maize and discusses this expression in relation to the developmental stage of the plant.
Background
Although genetic variation for protein content has been found in forage plants, this variability is narrower than that observed for other traits such as digestibility [ 1 ]. Since the major protein components in monocot forage and silage crops are involved in metabolic activity, and hence are not "true" storage proteins, it has been argued that it is not feasible to make major changes in protein quality or protein composition by conventional breeding [ 1 ]. However, genetic engineering may allow improvement in protein quality and content through expression of a storage protein not found in grass vegetative tissue.Genes encoding seed storage proteins of various plant species have been transgenically expressed to test for improvement of nutritional quality. Most experiments were conducted with tobacco and legume species including alfalfa, soybean, canola, clover and lupins. For nuclear-targeted genes, accumulation of these seed storage proteins in vegetative tissue of transgenic plants was either undetectable or very low. These included pea vicilin [ 2 , 3 ], soybean conglycinin [ 4 ], sunflower seed agglutinin [ 5 , 6 ], and phaseolin [ 7 ]. The instability of seed proteins in non-seed tissues of transgenic plants was frequently attributed to protein targeting to protease-rich vacuoles in the vegetative cells, and subsequent degradation [ 5 , 7 , 8 ].The greatest accumulation of a seed storage protein from a nuclear-targeted gene was achieved using zeins, a maize seed storage protein that is targeted to "protein bodies" directly from the endoplasmic reticulum (ER), thus avoiding the secretory route to the cellular vacuole. Transgenic expression from the CaMV 35S promoter in tobacco resulted in the formation of these protein bodies containing the zein within vegetative tissues [ 8 , 9 ]. Alternatively, "short-circuiting" the protein-targeting route by addition of an ER retention signal to the storage-protein coding region also increased protein accumulation up to 100× [ 5 , 6 , 10 , 11 ].In many legumes, accumulation of specific vegetative storage proteins (VSPs) in leaves and stems is the main source of increased nitrogen content [ 12 - 14 ]. Use of VSPs instead of seed storage proteins to increase vegetative protein content in monocots may provide an advantage since they have evolved to function in vegetative cell types. Legume vegetative cells that accumulate VSP proteins contain multiple vacuole types and storage proteins are targeted to specific vacuoles where they are not rapidly degraded [ 15 ], and do not interfere with cellular metabolic processes. It is uncertain if monocots can produce similar vacuoles or successfully target a VSP to them. The most studied VSPs are the soybean VSPα and VSPβ proteins, which are lysine-rich glycosylated vacuolar proteins that accumulate abundantly in leaves, stems and pods, but not in seeds [ 12 - 14 ]. Recently, soybean  vspA  and/or  vspB  genes fused to the 35S promoter were expressed in transgenic tobacco to study their accumulation in a heterologous dicot plant. Nuclear targeted genes produced VSP ranging between 2 and 6% of the soluble protein in leaves of the transgenic plants [ 16 ]; whereas, targeting to both the chloroplast and the vacuole within the same plant resulted in VSP comprising greater than 10% soluble protein [ 17 , 18 ]. Soybean VSP is therefore an excellent candidate for use in transgenic improvement of plant protein status, particularly grasses that contain limited levels of lysine [ 16 ]. However, it remains to be determined if VSPs can be expressed and accumulated in monocot plants since storage protein stability is dependent on post-translational events that may differ between monocots and eudicots. This manuscript presents the transgenic expression of soybean VSPβ in the leaves of transgenic maize and discusses this expression in relation to the developmental stage of the plant.vspAvspBResults and Discussion Development of primary (R 0 ) transgenic maize expressing  vspB Out of 101 plants, twenty, belonging to five independent lines (71-1, 45-1, 45-3, 44-1, and 4-1) were shown by Southern blot analysis to contain a 1.5 kb hybridizing band corresponding to the intact  bar  gene (Fig.  1 ). All 20 plants also contained the expected 1.9 kb band that hybridized to the  vspB  gene. The same probe detected two bands in  Eco RI restricted Soybean genomic DNA, ~5.7 and 8.6 Kb, corresponding to the highly homologous genes  vspA  and  vspB  [ 13 ]. Western blot analysis was used to detect the VSPβ polypeptide in leaf extracts from fourteen primary (R 0 ) transgenic maize plants at vegetative stage (7 weeks old). A distinct VSPβ band was not visible in silver-stained SDS-PAGE separated maize extract proteins due to complexity of the total protein pattern and the relatively low level of VSPβ expression (Fig.  2a ). However, some of these plants expressed VSPβ at a level high enough to be detected by Western blot analysis (Fig.  2b ). Two plants (71-1-53 and 45-3-1) had the highest level of VSPβ, while four plants (71-1-23, 71-1-20, 45-1-4, and 45-1-7) accumulated a lower level of VSPβ. Although a faint lower MW peptide band was visible in negative control maize extracts, VSP was clearly only present in transgenic lines. Faint detection of bands in untransformed maize (none of identical size to soybean VSPβ) is consistent with previous reports of cross-hybridizing of soybean VSP antibodies with proteins from monocots [ 14 ]. In soybean extracts used as a positive control, the antibody for VSPβ recognized both VSPα and VSPβ polypeptides (Fig.  2b,c ) Computer analysis of digital images of the Western blots was used to detect differences in relative band intensity of the immunologically detected VSPβ peptide. Because the native soybean VSPs (VSPα plus VSPβ) were easily visible on total protein stained gels (Fig.  2a ), relative quantification of total stained proteins in the soybean samples indicated that the VSPs represented about 10% of the total soluble protein in young soybean leaves. This is close to the previously reported value of 15 % [ 12 ]. The intensity of the transgenic maize 45-3-1 VSPβ band on Western blots was 44% of the soybean VSP's band (digital image pixel quantification of Fig.  2b ). Accounting for the differences in total protein applied to the gel (less soybean total protein was loaded), the VSPβ protein was estimated to have accumulated to 0.5 % of the total soluble protein. This is similar to the highest level of seed storage protein accumulation observed with the ectopic expression of zein [ 8 , 9 ], but remains less than the 1% minimal expression level predicted by Wandelt et al. [ 11 ] to be needed to directly alter the nutritional quality of the leaves. Although the 0.5% of total soluble protein was too low to alter nutritional value, detection of VSPβ in 45-3-1 allowed monitoring of VSPβ level in leaves and stems during plant development. Presence of  vspB  in R 1  plants R 1  plants were produced by back-crossing the R 0  plants with Hi II control non-transformed pollen. Back-crossing was performed because R 0  plants directly regenerated from tissue culture did not have synchronized production of pollen and receptive female flowers. The R 1  families segregating for  bar  expression were analyzed for the presence of the  vspB  gene by Southern blot analysis. From 57 R 1  plants analyzed, Southern blot analysis showed that 35 (61%) contained the  vspB  gene integrated into the genome. Figure  3  shows examples for several R 1  from five R 0  parental lines. The ~1.9 kb  vspB  band can be seen in nine of the 16 R 1  lines. There are also fainter bands one slightly larger than the 1.9 kb band and one or two migrating between 4 and 5 kb. These are often observed as incomplete restriction of all  Eco RI  sites internal to plasmid DNA that is integrated into the maize genome. Similar bands are observed even with the plasmid control. Total RNA was isolated from leaves of young R 1  plants at the vegetative stage (7 weeks after planting). Eighteen transgenic R 1  plants, including the ones originating from R 0  plants representing different levels of VSPβ accumulation (high VSPβ accumulators: 45-3-1, 71-1-53; mid-level VSPβ accumulators: and 71-1-23, 71-1-20, 45-1-4, 45-1-7 and low VSPβ accumulators: 44-1-1- 4-1-2), were analyzed for  vspB  transcripts. Because of high sequence similarity (85%) between  vspA  and  vspB  cDNAs, the same probe hybridized in soybean with both mRNAs as demonstrated by Staswick [ 13 ]. The transgenic plants 71-1-53A, 71-1-20A, 71-1-20E, and 71-1-20I produced a hybridizing band of approximately the expected size of 1.1 kb indicating transgene expression (Fig.  4a ); however, most did not. Use of the  Ubi -1 promoter for both the  vspB  and  bar  gene probably led to a high number of transgenics with the  vspB  silenced, and a higher level of VSPβ accumulation will likely result in future work using a combination of different promoters. Immunodetection of VSPβ protein in transgenic young plant leaves showed variation in accumulation in comparisons between the parental (R 0 ) and their R 1  progeny, with the greatest variation observed with the highest VSPβ expressing R 0  plants (Fig  4b ). The parental line 71-1-53 had a relatively high level of expression of VSPβ, but the only transgenic offspring from this line, 71-1-53A, had almost undetectable VSPβ; in contrast, a low level accumulator, 71-1-20, produced R 1  lines expressing different levels of VSPβ, although none of them expressed at higher levels than the parent. Quantification of the relative level of VSPβ expressed in the R 1  leaves showed that the highest level measured was only 0.03% of total soluble protein. Despite the overall low level of VSPβ expression, for the purposes of this work, VSPβ accumulation in several of the plants was high enough to study the relationship of plant developmental stage and  vspB /VSPβ accumulation. Both the transcript abundance and VSPβ protein accumulation were determined in the R 1  lines using real-time quantitative RT-PCR and Western blot immunodetection, respectively. Real-time RT-PCR is more sensitive that Northern blot analysis and was able to detect transcripts that were not seen with standard total RNA blotting methods. The  vspB  transcript was quantified in leaves from immature plants (prior to tassel formation) and silage stage plants (plants with developing seeds at the 18 DAP-days after pollination stage), as well as, stems from the silage stage plants, (Figure  5 ). The  vspB  transcript was detected in all five transgenics (four of which had RNA not detectable using standard Northern blot methods). The relative level of RNA among the different plant samples was not consistent across the different lines with some having more transcripts in the young leaves while others had more in the older leaves and stems. When  vspB  RNA abundance was compared to VSPβ protein accumulation there was no correlation between the two (Fig.  5a, b, c ). Antiserum to VSPβ detected VSPβ only in leaves from young maize plants that had not yet flowered. No VSPβ was detected in leaves and stems of silage stage corn that had developing seed. The soybean VSPβ peptide was the primary band reacting with the antiVSPβ antiserum in young leaves of transgenic maize, however, in silage stage stems and to a lesser extent the silage stage leaves, there were multiple bands detected at a different size than the VSPβ. These were also detected in the non-transgenic control plant samples and therefore, were not due to different modifications of the soybean VSPβ. Although the VSPβ protein dropped below detectable levels in the silage stage 71-1-20A, 45-3-1F and 45-3-1-G plants, the  vspβ  transcript was still detectable. In fact, in the 71-1-20A plants the transcript level was highest in the silage stage leaves that had no detectable VSPβ. Therefore, post-transcriptional events (i.e., changes in either the translational efficiency of the  vspB  transcript or the protein stability of VSPβ) were altered in the silage stage leaves and stems as compared to the leaves of immature plants.
Results and Discussion
Development of primary (R 0 ) transgenic maize expressing  vspB Out of 101 plants, twenty, belonging to five independent lines (71-1, 45-1, 45-3, 44-1, and 4-1) were shown by Southern blot analysis to contain a 1.5 kb hybridizing band corresponding to the intact  bar  gene (Fig.  1 ). All 20 plants also contained the expected 1.9 kb band that hybridized to the  vspB  gene. The same probe detected two bands in  Eco RI restricted Soybean genomic DNA, ~5.7 and 8.6 Kb, corresponding to the highly homologous genes  vspA  and  vspB  [ 13 ]. Western blot analysis was used to detect the VSPβ polypeptide in leaf extracts from fourteen primary (R 0 ) transgenic maize plants at vegetative stage (7 weeks old). A distinct VSPβ band was not visible in silver-stained SDS-PAGE separated maize extract proteins due to complexity of the total protein pattern and the relatively low level of VSPβ expression (Fig.  2a ). However, some of these plants expressed VSPβ at a level high enough to be detected by Western blot analysis (Fig.  2b ). Two plants (71-1-53 and 45-3-1) had the highest level of VSPβ, while four plants (71-1-23, 71-1-20, 45-1-4, and 45-1-7) accumulated a lower level of VSPβ. Although a faint lower MW peptide band was visible in negative control maize extracts, VSP was clearly only present in transgenic lines. Faint detection of bands in untransformed maize (none of identical size to soybean VSPβ) is consistent with previous reports of cross-hybridizing of soybean VSP antibodies with proteins from monocots [ 14 ]. In soybean extracts used as a positive control, the antibody for VSPβ recognized both VSPα and VSPβ polypeptides (Fig.  2b,c ) Computer analysis of digital images of the Western blots was used to detect differences in relative band intensity of the immunologically detected VSPβ peptide. Because the native soybean VSPs (VSPα plus VSPβ) were easily visible on total protein stained gels (Fig.  2a ), relative quantification of total stained proteins in the soybean samples indicated that the VSPs represented about 10% of the total soluble protein in young soybean leaves. This is close to the previously reported value of 15 % [ 12 ]. The intensity of the transgenic maize 45-3-1 VSPβ band on Western blots was 44% of the soybean VSP's band (digital image pixel quantification of Fig.  2b ). Accounting for the differences in total protein applied to the gel (less soybean total protein was loaded), the VSPβ protein was estimated to have accumulated to 0.5 % of the total soluble protein. This is similar to the highest level of seed storage protein accumulation observed with the ectopic expression of zein [ 8 , 9 ], but remains less than the 1% minimal expression level predicted by Wandelt et al. [ 11 ] to be needed to directly alter the nutritional quality of the leaves. Although the 0.5% of total soluble protein was too low to alter nutritional value, detection of VSPβ in 45-3-1 allowed monitoring of VSPβ level in leaves and stems during plant development.
Development of primary (R 0 ) transgenic maize expressing  vspB
vspBOut of 101 plants, twenty, belonging to five independent lines (71-1, 45-1, 45-3, 44-1, and 4-1) were shown by Southern blot analysis to contain a 1.5 kb hybridizing band corresponding to the intact  bar  gene (Fig.  1 ). All 20 plants also contained the expected 1.9 kb band that hybridized to the  vspB  gene. The same probe detected two bands in  Eco RI restricted Soybean genomic DNA, ~5.7 and 8.6 Kb, corresponding to the highly homologous genes  vspA  and  vspB  [ 13 ].barvspBEcovspAvspBWestern blot analysis was used to detect the VSPβ polypeptide in leaf extracts from fourteen primary (R 0 ) transgenic maize plants at vegetative stage (7 weeks old). A distinct VSPβ band was not visible in silver-stained SDS-PAGE separated maize extract proteins due to complexity of the total protein pattern and the relatively low level of VSPβ expression (Fig.  2a ). However, some of these plants expressed VSPβ at a level high enough to be detected by Western blot analysis (Fig.  2b ). Two plants (71-1-53 and 45-3-1) had the highest level of VSPβ, while four plants (71-1-23, 71-1-20, 45-1-4, and 45-1-7) accumulated a lower level of VSPβ. Although a faint lower MW peptide band was visible in negative control maize extracts, VSP was clearly only present in transgenic lines. Faint detection of bands in untransformed maize (none of identical size to soybean VSPβ) is consistent with previous reports of cross-hybridizing of soybean VSP antibodies with proteins from monocots [ 14 ]. In soybean extracts used as a positive control, the antibody for VSPβ recognized both VSPα and VSPβ polypeptides (Fig.  2b,c )Computer analysis of digital images of the Western blots was used to detect differences in relative band intensity of the immunologically detected VSPβ peptide. Because the native soybean VSPs (VSPα plus VSPβ) were easily visible on total protein stained gels (Fig.  2a ), relative quantification of total stained proteins in the soybean samples indicated that the VSPs represented about 10% of the total soluble protein in young soybean leaves. This is close to the previously reported value of 15 % [ 12 ]. The intensity of the transgenic maize 45-3-1 VSPβ band on Western blots was 44% of the soybean VSP's band (digital image pixel quantification of Fig.  2b ). Accounting for the differences in total protein applied to the gel (less soybean total protein was loaded), the VSPβ protein was estimated to have accumulated to 0.5 % of the total soluble protein. This is similar to the highest level of seed storage protein accumulation observed with the ectopic expression of zein [ 8 , 9 ], but remains less than the 1% minimal expression level predicted by Wandelt et al. [ 11 ] to be needed to directly alter the nutritional quality of the leaves. Although the 0.5% of total soluble protein was too low to alter nutritional value, detection of VSPβ in 45-3-1 allowed monitoring of VSPβ level in leaves and stems during plant development.Presence of  vspB  in R 1  plants R 1  plants were produced by back-crossing the R 0  plants with Hi II control non-transformed pollen. Back-crossing was performed because R 0  plants directly regenerated from tissue culture did not have synchronized production of pollen and receptive female flowers. The R 1  families segregating for  bar  expression were analyzed for the presence of the  vspB  gene by Southern blot analysis. From 57 R 1  plants analyzed, Southern blot analysis showed that 35 (61%) contained the  vspB  gene integrated into the genome. Figure  3  shows examples for several R 1  from five R 0  parental lines. The ~1.9 kb  vspB  band can be seen in nine of the 16 R 1  lines. There are also fainter bands one slightly larger than the 1.9 kb band and one or two migrating between 4 and 5 kb. These are often observed as incomplete restriction of all  Eco RI  sites internal to plasmid DNA that is integrated into the maize genome. Similar bands are observed even with the plasmid control. Total RNA was isolated from leaves of young R 1  plants at the vegetative stage (7 weeks after planting). Eighteen transgenic R 1  plants, including the ones originating from R 0  plants representing different levels of VSPβ accumulation (high VSPβ accumulators: 45-3-1, 71-1-53; mid-level VSPβ accumulators: and 71-1-23, 71-1-20, 45-1-4, 45-1-7 and low VSPβ accumulators: 44-1-1- 4-1-2), were analyzed for  vspB  transcripts. Because of high sequence similarity (85%) between  vspA  and  vspB  cDNAs, the same probe hybridized in soybean with both mRNAs as demonstrated by Staswick [ 13 ]. The transgenic plants 71-1-53A, 71-1-20A, 71-1-20E, and 71-1-20I produced a hybridizing band of approximately the expected size of 1.1 kb indicating transgene expression (Fig.  4a ); however, most did not. Use of the  Ubi -1 promoter for both the  vspB  and  bar  gene probably led to a high number of transgenics with the  vspB  silenced, and a higher level of VSPβ accumulation will likely result in future work using a combination of different promoters. Immunodetection of VSPβ protein in transgenic young plant leaves showed variation in accumulation in comparisons between the parental (R 0 ) and their R 1  progeny, with the greatest variation observed with the highest VSPβ expressing R 0  plants (Fig  4b ). The parental line 71-1-53 had a relatively high level of expression of VSPβ, but the only transgenic offspring from this line, 71-1-53A, had almost undetectable VSPβ; in contrast, a low level accumulator, 71-1-20, produced R 1  lines expressing different levels of VSPβ, although none of them expressed at higher levels than the parent. Quantification of the relative level of VSPβ expressed in the R 1  leaves showed that the highest level measured was only 0.03% of total soluble protein. Despite the overall low level of VSPβ expression, for the purposes of this work, VSPβ accumulation in several of the plants was high enough to study the relationship of plant developmental stage and  vspB /VSPβ accumulation. Both the transcript abundance and VSPβ protein accumulation were determined in the R 1  lines using real-time quantitative RT-PCR and Western blot immunodetection, respectively. Real-time RT-PCR is more sensitive that Northern blot analysis and was able to detect transcripts that were not seen with standard total RNA blotting methods. The  vspB  transcript was quantified in leaves from immature plants (prior to tassel formation) and silage stage plants (plants with developing seeds at the 18 DAP-days after pollination stage), as well as, stems from the silage stage plants, (Figure  5 ). The  vspB  transcript was detected in all five transgenics (four of which had RNA not detectable using standard Northern blot methods). The relative level of RNA among the different plant samples was not consistent across the different lines with some having more transcripts in the young leaves while others had more in the older leaves and stems. When  vspB  RNA abundance was compared to VSPβ protein accumulation there was no correlation between the two (Fig.  5a, b, c ). Antiserum to VSPβ detected VSPβ only in leaves from young maize plants that had not yet flowered. No VSPβ was detected in leaves and stems of silage stage corn that had developing seed. The soybean VSPβ peptide was the primary band reacting with the antiVSPβ antiserum in young leaves of transgenic maize, however, in silage stage stems and to a lesser extent the silage stage leaves, there were multiple bands detected at a different size than the VSPβ. These were also detected in the non-transgenic control plant samples and therefore, were not due to different modifications of the soybean VSPβ. Although the VSPβ protein dropped below detectable levels in the silage stage 71-1-20A, 45-3-1F and 45-3-1-G plants, the  vspβ  transcript was still detectable. In fact, in the 71-1-20A plants the transcript level was highest in the silage stage leaves that had no detectable VSPβ. Therefore, post-transcriptional events (i.e., changes in either the translational efficiency of the  vspB  transcript or the protein stability of VSPβ) were altered in the silage stage leaves and stems as compared to the leaves of immature plants.
Presence of  vspB  in R 1  plants
vspBR 1  plants were produced by back-crossing the R 0  plants with Hi II control non-transformed pollen. Back-crossing was performed because R 0  plants directly regenerated from tissue culture did not have synchronized production of pollen and receptive female flowers. The R 1  families segregating for  bar  expression were analyzed for the presence of the  vspB  gene by Southern blot analysis. From 57 R 1  plants analyzed, Southern blot analysis showed that 35 (61%) contained the  vspB  gene integrated into the genome. Figure  3  shows examples for several R 1  from five R 0  parental lines. The ~1.9 kb  vspB  band can be seen in nine of the 16 R 1  lines. There are also fainter bands one slightly larger than the 1.9 kb band and one or two migrating between 4 and 5 kb. These are often observed as incomplete restriction of all  Eco RI  sites internal to plasmid DNA that is integrated into the maize genome. Similar bands are observed even with the plasmid control.barvspBvspBvspBEco RITotal RNA was isolated from leaves of young R 1  plants at the vegetative stage (7 weeks after planting). Eighteen transgenic R 1  plants, including the ones originating from R 0  plants representing different levels of VSPβ accumulation (high VSPβ accumulators: 45-3-1, 71-1-53; mid-level VSPβ accumulators: and 71-1-23, 71-1-20, 45-1-4, 45-1-7 and low VSPβ accumulators: 44-1-1- 4-1-2), were analyzed for  vspB  transcripts. Because of high sequence similarity (85%) between  vspA  and  vspB  cDNAs, the same probe hybridized in soybean with both mRNAs as demonstrated by Staswick [ 13 ]. The transgenic plants 71-1-53A, 71-1-20A, 71-1-20E, and 71-1-20I produced a hybridizing band of approximately the expected size of 1.1 kb indicating transgene expression (Fig.  4a ); however, most did not. Use of the  Ubi -1 promoter for both the  vspB  and  bar  gene probably led to a high number of transgenics with the  vspB  silenced, and a higher level of VSPβ accumulation will likely result in future work using a combination of different promoters.vspBvspAvspBUbivspBbarvspBImmunodetection of VSPβ protein in transgenic young plant leaves showed variation in accumulation in comparisons between the parental (R 0 ) and their R 1  progeny, with the greatest variation observed with the highest VSPβ expressing R 0  plants (Fig  4b ). The parental line 71-1-53 had a relatively high level of expression of VSPβ, but the only transgenic offspring from this line, 71-1-53A, had almost undetectable VSPβ; in contrast, a low level accumulator, 71-1-20, produced R 1  lines expressing different levels of VSPβ, although none of them expressed at higher levels than the parent. Quantification of the relative level of VSPβ expressed in the R 1  leaves showed that the highest level measured was only 0.03% of total soluble protein.Despite the overall low level of VSPβ expression, for the purposes of this work, VSPβ accumulation in several of the plants was high enough to study the relationship of plant developmental stage and  vspB /VSPβ accumulation. Both the transcript abundance and VSPβ protein accumulation were determined in the R 1  lines using real-time quantitative RT-PCR and Western blot immunodetection, respectively. Real-time RT-PCR is more sensitive that Northern blot analysis and was able to detect transcripts that were not seen with standard total RNA blotting methods. The  vspB  transcript was quantified in leaves from immature plants (prior to tassel formation) and silage stage plants (plants with developing seeds at the 18 DAP-days after pollination stage), as well as, stems from the silage stage plants, (Figure  5 ). The  vspB  transcript was detected in all five transgenics (four of which had RNA not detectable using standard Northern blot methods). The relative level of RNA among the different plant samples was not consistent across the different lines with some having more transcripts in the young leaves while others had more in the older leaves and stems.vspBvspBvspBWhen  vspB  RNA abundance was compared to VSPβ protein accumulation there was no correlation between the two (Fig.  5a, b, c ). Antiserum to VSPβ detected VSPβ only in leaves from young maize plants that had not yet flowered. No VSPβ was detected in leaves and stems of silage stage corn that had developing seed. The soybean VSPβ peptide was the primary band reacting with the antiVSPβ antiserum in young leaves of transgenic maize, however, in silage stage stems and to a lesser extent the silage stage leaves, there were multiple bands detected at a different size than the VSPβ. These were also detected in the non-transgenic control plant samples and therefore, were not due to different modifications of the soybean VSPβ. Although the VSPβ protein dropped below detectable levels in the silage stage 71-1-20A, 45-3-1F and 45-3-1-G plants, the  vspβ  transcript was still detectable. In fact, in the 71-1-20A plants the transcript level was highest in the silage stage leaves that had no detectable VSPβ. Therefore, post-transcriptional events (i.e., changes in either the translational efficiency of the  vspB  transcript or the protein stability of VSPβ) were altered in the silage stage leaves and stems as compared to the leaves of immature plants.vspBvspβvspBConclusion The  vspB  gene was successfully introduced into R 0  regenerated maize and transferred to the R 1  progeny, of which  vspB  transcript and VSPβ protein were detected and studied. This is the first report on introduction and expression of a legume vegetative storage protein in a monocot plant. The inability to detect VSPβ from the maize vegetative tissue at the time of seed development, even when the  vspB  transcript was still expressed, must have arisen from either reduced translational efficiency of the  vspB  transcript or a decrease in the stability of the VSPβ protein. The reduction of seed storage protein level in leaves of transgenic eudicots was also observed with expression of vicillin in alfalfa [ 11 ] and tobacco [ 2 ]; however, it was not observed with the expression of VSPα in tobacco [ 16 ] or ovalbumin in alfalfa [ 30 ]. These data suggest that factors controlling developmental change in vegetative tissue protein accumulation are a combination of host plant traits and innate characteristics of the ectopically expressed protein. It is interesting to speculate that if, in maize (a monocot), soybean VSPβ was degraded in a manner that provided amino acids that were translocated to the seed to support seed development, then development of high level VSP expressing monocots may be a way to improve nitrogen content of the seed/grain produced by the plant.
Conclusion
The  vspB  gene was successfully introduced into R 0  regenerated maize and transferred to the R 1  progeny, of which  vspB  transcript and VSPβ protein were detected and studied. This is the first report on introduction and expression of a legume vegetative storage protein in a monocot plant. The inability to detect VSPβ from the maize vegetative tissue at the time of seed development, even when the  vspB  transcript was still expressed, must have arisen from either reduced translational efficiency of the  vspB  transcript or a decrease in the stability of the VSPβ protein. The reduction of seed storage protein level in leaves of transgenic eudicots was also observed with expression of vicillin in alfalfa [ 11 ] and tobacco [ 2 ]; however, it was not observed with the expression of VSPα in tobacco [ 16 ] or ovalbumin in alfalfa [ 30 ]. These data suggest that factors controlling developmental change in vegetative tissue protein accumulation are a combination of host plant traits and innate characteristics of the ectopically expressed protein. It is interesting to speculate that if, in maize (a monocot), soybean VSPβ was degraded in a manner that provided amino acids that were translocated to the seed to support seed development, then development of high level VSP expressing monocots may be a way to improve nitrogen content of the seed/grain produced by the plant.vspBvspBvspBvspBMethods Plant material and tissue culture methods Plants of hybrid "Hi-II" maize were established in a greenhouse and immature tassels were used for embryogenic type II callus production, as described by Armstrong [ 19 ]. Transgenic plant development Microprojectile bombardment of callus was performed using the procedure of Somers et al. [ 20 ]. Calli were cobombarded with equal amounts of pRSVP-1 (Shatters Jr., unpublished) and pAHC25 [ 21 ]. Plasmid pRSVP-1 was constructed by restricting the soybean  vspB  cDNA clone (998 bp) from pKSH3 [ 22 ] with  Eco RI, blunt ends of this fragment were produced using S1 nuclease and the fragment was cloned into similarly blunt ended  Bam HI restricted pAHC17 [ 21 ]. As a result, the  vspB  coding region was inserted downstream of the  Ubi-1  promoter and a 5' untranslated region (exon) and intron; and upstream of the  nos  terminator sequence. The plasmid pAHC25 carried the  bar  gene and the  uidA  reporter gene, both under the control of the  Ubi-1  promoter. Bombardments were performed with a Biolistic ®  PDS-1000/He Particle Delivery System (Bio-Rad Laboratories, Hercules, CA) and an osmotic treatment was applied to reduce the cell damage caused by the gene transfer method [ 23 ]. Putative transgenic maize were regenerated from glufosinate resistant callus as described by Armstrong [ 19 ], and grown in five-gallon pots containing sterile sand and Metromix-350 (1:1). Plants were fertilized weekly with Peter's 20-20-20 with micronutrients (Division of United Industry Corp., St. Louis, MO). Southern blot analysis One gram of frozen young leaf tissue was ground in liquid nitrogen and genomic DNA was extracted using the Dellaporta procedure [ 24 ]. Twenty micrograms of genomic DNA were digested with  Eco RI, which released a 1.9 Kb fragment containing the  vspB  gene, the  nos  terminator and part of the  Ubi-1  promoter. DNA was separated on a 0.8% agarose gel, blotted onto Hybond N +  membrane (Amersham Pharmacia Biotech, Inc. Piscataway, NJ) by capillary blotting [ 25 ], and UV cross-linked. The non-radioactive digoxigenin system (Roche Molecular Biochemicals, Indianapolis, IN) was used for labeling and detection of the transgene. Blotted DNA was probed with either a 611 or a 843 bp of  vspB  gene segment amplified from pRSVP-1 and gel purified. The forward and reverse primers 5'-GTTCTTCGGAG GTAAAAT-3' and 5'-TTCGCCTCTGTGGT-3' were used, respectively, to amplify a 611 bp segment, and the primer pair 5'-GCAGGCTACCAAAGGT-3' and 5'-TAGGTGACTTACCCACAT-3' was used to amplified the product of 843 bp. For identification of  bar  transgenic plants, the DNA was digested with  Eco RI, which released a fragment of ~1.5 Kb that contained part of the  Ubi-1  promoter, the  bar  gene, and the  nos  terminator, and was identified with a 419 bp digoxigenin labeled probe produced by PCR amplification of pAHC25 using the forward and reverse primers: 5'-GGCGGTCTGCACCATCGT-3' and 5'-GCCAAGTTCCCGTGCTTGA-3', respectively. Northern blot analysis Total RNA was isolated from 2 g of tissue using acid guanidine isothiocyanate-phenol-chlorophorm extraction [ 26 ], resuspended in T 10 E 1  and treated with 2 μl RNasin ®  (4U/μl) RNAse inhibitor (Promega, Madison, WI) and stored at -70°C until use. Thirty micrograms of total RNA were separated on 1.2% agarose formaldehyde gels and transferred to Hybond N +  membrane by capillary blotting overnight or by pressure blotting for one hour using a PosiBlot ®  30-30 pressure blotter (Stratagene, La Jolla, CA) according to the manufacture's instructions. Membranes were UV-crosslinked, and probed with the same probes as described for Southern blot analysis. Chemilluminescence was captured using a Kodak Digital Science Image Station 440 CF (Eastman Kodak Company, Rochester, NY). Real-Time RT-PCR Total RNA extractions for real-time RT-PCR were performed using 500 mg of tissue ground to fine powder using mortar and pestle in the presence of liquid nitrogen, then processed with the RNeasy midiprep Kit (Qiagen, Germany), following the manufacturer's protocol. Trace DNA contamination was removed from total RNA by a combination of acid phenol: chloroform 5:1 pH= 4.7 extraction and Dnase I treatment (Ambion, Texas). Real-time RT-PCR was performed on a Rotor-Gene RG-3000 (Corbett Research, Australia) using the Quantitect SYBR Green real-time RT-PCR kit (Qiagen, Germany), and the manufacturers protocols with 300 ng of Dnase I treated total RNA. Primers were designed to amplify a 108 bp fragment of the soybean  vspB  using the following primers: 5'-TGGTTCAACGCACTCTTC-3' and 5'-GGCTATGGTGAGCGTTCTTC-3'. Reverse transcription was performed for 30 min at 50°C followed by a 15 min denaturing at 95°C, and 40 cycles of 40 s at 95°C, 40 s at 58°C and 40 s at 72°C. Quantification was based on relative abundance to maize 18S RNA by amplifying a 174 bp fragment with primers: 5'-CCTGCGGCTTAATTGACTC-3' and 5'-GTTAGCAGGCTGAGGTCTCG-3', and using the comparative quantification function of the Rotor-Gene RG-3000 software. All real-time RT-PCR experiments were conducted in triplicate and on triplicate RNA preparations for each sample. Melting curve analysis and agarose gel electrophoresis were performed to verify single product formation. Western blot analysis Protein was extracted from 100 mg of leaves and stems with 0.5 ml of phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 ) supplemented with 1 tablet/10 ml buffer of the Complete-Mini protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN) by homogenizing in the presence of zirconia/silica 0.1 mm dia. beads. Centrifuged extract supernatants were removed and used for protein concentration determination [ 27 ]. Because of the low protein yields in stem extracts, they were precipitated with 10% trichloroacetic acid (TCA), washed with ice-cold acetone and resuspended prior to SDS-PAGE analysis. Thirty micrograms of protein were separated on 12% SDS-polyacrylamide gels. Proteins in the gel were either stained with silver nitrate [ 28 ] or transferred to Hybond-P (PVDF) membranes (Amersham Pharmacia Biotech, Inc. Piscataway, NJ) using Trans-Blot SD Semi-Dry Transfer Cell blotter and recommended protocols (Bio-Rad Laboratories). Soybean VSP was immunologically detected on the PDF membranes with anti VSPβ serum (provided by P. Staswick, University of Nebraska, produced as previously described [ 29 ] used at a 1:5,000 dilution. Detection was performed using a luminol substrate and the NEN Life Science Products, Inc. (Boston, MA) Reinascence kit. Chemiluminescent signal was captured by a Kodak Digital Science Image Station 440 Cf, and analyzed with Kodak 1D Scientific Image Software. Quantification of band intensity was always compared relative to samples from the same gel.
Methods
Plant material and tissue culture methods Plants of hybrid "Hi-II" maize were established in a greenhouse and immature tassels were used for embryogenic type II callus production, as described by Armstrong [ 19 ].
Plant material and tissue culture methods
Plants of hybrid "Hi-II" maize were established in a greenhouse and immature tassels were used for embryogenic type II callus production, as described by Armstrong [ 19 ].Transgenic plant development Microprojectile bombardment of callus was performed using the procedure of Somers et al. [ 20 ]. Calli were cobombarded with equal amounts of pRSVP-1 (Shatters Jr., unpublished) and pAHC25 [ 21 ]. Plasmid pRSVP-1 was constructed by restricting the soybean  vspB  cDNA clone (998 bp) from pKSH3 [ 22 ] with  Eco RI, blunt ends of this fragment were produced using S1 nuclease and the fragment was cloned into similarly blunt ended  Bam HI restricted pAHC17 [ 21 ]. As a result, the  vspB  coding region was inserted downstream of the  Ubi-1  promoter and a 5' untranslated region (exon) and intron; and upstream of the  nos  terminator sequence. The plasmid pAHC25 carried the  bar  gene and the  uidA  reporter gene, both under the control of the  Ubi-1  promoter. Bombardments were performed with a Biolistic ®  PDS-1000/He Particle Delivery System (Bio-Rad Laboratories, Hercules, CA) and an osmotic treatment was applied to reduce the cell damage caused by the gene transfer method [ 23 ]. Putative transgenic maize were regenerated from glufosinate resistant callus as described by Armstrong [ 19 ], and grown in five-gallon pots containing sterile sand and Metromix-350 (1:1). Plants were fertilized weekly with Peter's 20-20-20 with micronutrients (Division of United Industry Corp., St. Louis, MO).
Transgenic plant development
Microprojectile bombardment of callus was performed using the procedure of Somers et al. [ 20 ]. Calli were cobombarded with equal amounts of pRSVP-1 (Shatters Jr., unpublished) and pAHC25 [ 21 ]. Plasmid pRSVP-1 was constructed by restricting the soybean  vspB  cDNA clone (998 bp) from pKSH3 [ 22 ] with  Eco RI, blunt ends of this fragment were produced using S1 nuclease and the fragment was cloned into similarly blunt ended  Bam HI restricted pAHC17 [ 21 ]. As a result, the  vspB  coding region was inserted downstream of the  Ubi-1  promoter and a 5' untranslated region (exon) and intron; and upstream of the  nos  terminator sequence. The plasmid pAHC25 carried the  bar  gene and the  uidA  reporter gene, both under the control of the  Ubi-1  promoter. Bombardments were performed with a Biolistic ®  PDS-1000/He Particle Delivery System (Bio-Rad Laboratories, Hercules, CA) and an osmotic treatment was applied to reduce the cell damage caused by the gene transfer method [ 23 ]. Putative transgenic maize were regenerated from glufosinate resistant callus as described by Armstrong [ 19 ], and grown in five-gallon pots containing sterile sand and Metromix-350 (1:1). Plants were fertilized weekly with Peter's 20-20-20 with micronutrients (Division of United Industry Corp., St. Louis, MO).vspBEcoBamvspBUbi-1nosbaruidAUbi-1Southern blot analysis One gram of frozen young leaf tissue was ground in liquid nitrogen and genomic DNA was extracted using the Dellaporta procedure [ 24 ]. Twenty micrograms of genomic DNA were digested with  Eco RI, which released a 1.9 Kb fragment containing the  vspB  gene, the  nos  terminator and part of the  Ubi-1  promoter. DNA was separated on a 0.8% agarose gel, blotted onto Hybond N +  membrane (Amersham Pharmacia Biotech, Inc. Piscataway, NJ) by capillary blotting [ 25 ], and UV cross-linked. The non-radioactive digoxigenin system (Roche Molecular Biochemicals, Indianapolis, IN) was used for labeling and detection of the transgene. Blotted DNA was probed with either a 611 or a 843 bp of  vspB  gene segment amplified from pRSVP-1 and gel purified. The forward and reverse primers 5'-GTTCTTCGGAG GTAAAAT-3' and 5'-TTCGCCTCTGTGGT-3' were used, respectively, to amplify a 611 bp segment, and the primer pair 5'-GCAGGCTACCAAAGGT-3' and 5'-TAGGTGACTTACCCACAT-3' was used to amplified the product of 843 bp. For identification of  bar  transgenic plants, the DNA was digested with  Eco RI, which released a fragment of ~1.5 Kb that contained part of the  Ubi-1  promoter, the  bar  gene, and the  nos  terminator, and was identified with a 419 bp digoxigenin labeled probe produced by PCR amplification of pAHC25 using the forward and reverse primers: 5'-GGCGGTCTGCACCATCGT-3' and 5'-GCCAAGTTCCCGTGCTTGA-3', respectively.
Southern blot analysis
One gram of frozen young leaf tissue was ground in liquid nitrogen and genomic DNA was extracted using the Dellaporta procedure [ 24 ]. Twenty micrograms of genomic DNA were digested with  Eco RI, which released a 1.9 Kb fragment containing the  vspB  gene, the  nos  terminator and part of the  Ubi-1  promoter. DNA was separated on a 0.8% agarose gel, blotted onto Hybond N +  membrane (Amersham Pharmacia Biotech, Inc. Piscataway, NJ) by capillary blotting [ 25 ], and UV cross-linked. The non-radioactive digoxigenin system (Roche Molecular Biochemicals, Indianapolis, IN) was used for labeling and detection of the transgene. Blotted DNA was probed with either a 611 or a 843 bp of  vspB  gene segment amplified from pRSVP-1 and gel purified. The forward and reverse primers 5'-GTTCTTCGGAG GTAAAAT-3' and 5'-TTCGCCTCTGTGGT-3' were used, respectively, to amplify a 611 bp segment, and the primer pair 5'-GCAGGCTACCAAAGGT-3' and 5'-TAGGTGACTTACCCACAT-3' was used to amplified the product of 843 bp.EcovspBnosUbi-1vspBFor identification of  bar  transgenic plants, the DNA was digested with  Eco RI, which released a fragment of ~1.5 Kb that contained part of the  Ubi-1  promoter, the  bar  gene, and the  nos  terminator, and was identified with a 419 bp digoxigenin labeled probe produced by PCR amplification of pAHC25 using the forward and reverse primers: 5'-GGCGGTCTGCACCATCGT-3' and 5'-GCCAAGTTCCCGTGCTTGA-3', respectively.barEcoUbi-1barnosNorthern blot analysis Total RNA was isolated from 2 g of tissue using acid guanidine isothiocyanate-phenol-chlorophorm extraction [ 26 ], resuspended in T 10 E 1  and treated with 2 μl RNasin ®  (4U/μl) RNAse inhibitor (Promega, Madison, WI) and stored at -70°C until use. Thirty micrograms of total RNA were separated on 1.2% agarose formaldehyde gels and transferred to Hybond N +  membrane by capillary blotting overnight or by pressure blotting for one hour using a PosiBlot ®  30-30 pressure blotter (Stratagene, La Jolla, CA) according to the manufacture's instructions. Membranes were UV-crosslinked, and probed with the same probes as described for Southern blot analysis. Chemilluminescence was captured using a Kodak Digital Science Image Station 440 CF (Eastman Kodak Company, Rochester, NY).
Northern blot analysis
Total RNA was isolated from 2 g of tissue using acid guanidine isothiocyanate-phenol-chlorophorm extraction [ 26 ], resuspended in T 10 E 1  and treated with 2 μl RNasin ®  (4U/μl) RNAse inhibitor (Promega, Madison, WI) and stored at -70°C until use. Thirty micrograms of total RNA were separated on 1.2% agarose formaldehyde gels and transferred to Hybond N +  membrane by capillary blotting overnight or by pressure blotting for one hour using a PosiBlot ®  30-30 pressure blotter (Stratagene, La Jolla, CA) according to the manufacture's instructions. Membranes were UV-crosslinked, and probed with the same probes as described for Southern blot analysis. Chemilluminescence was captured using a Kodak Digital Science Image Station 440 CF (Eastman Kodak Company, Rochester, NY).Real-Time RT-PCR Total RNA extractions for real-time RT-PCR were performed using 500 mg of tissue ground to fine powder using mortar and pestle in the presence of liquid nitrogen, then processed with the RNeasy midiprep Kit (Qiagen, Germany), following the manufacturer's protocol. Trace DNA contamination was removed from total RNA by a combination of acid phenol: chloroform 5:1 pH= 4.7 extraction and Dnase I treatment (Ambion, Texas). Real-time RT-PCR was performed on a Rotor-Gene RG-3000 (Corbett Research, Australia) using the Quantitect SYBR Green real-time RT-PCR kit (Qiagen, Germany), and the manufacturers protocols with 300 ng of Dnase I treated total RNA. Primers were designed to amplify a 108 bp fragment of the soybean  vspB  using the following primers: 5'-TGGTTCAACGCACTCTTC-3' and 5'-GGCTATGGTGAGCGTTCTTC-3'. Reverse transcription was performed for 30 min at 50°C followed by a 15 min denaturing at 95°C, and 40 cycles of 40 s at 95°C, 40 s at 58°C and 40 s at 72°C. Quantification was based on relative abundance to maize 18S RNA by amplifying a 174 bp fragment with primers: 5'-CCTGCGGCTTAATTGACTC-3' and 5'-GTTAGCAGGCTGAGGTCTCG-3', and using the comparative quantification function of the Rotor-Gene RG-3000 software. All real-time RT-PCR experiments were conducted in triplicate and on triplicate RNA preparations for each sample. Melting curve analysis and agarose gel electrophoresis were performed to verify single product formation.
Real-Time RT-PCR
Total RNA extractions for real-time RT-PCR were performed using 500 mg of tissue ground to fine powder using mortar and pestle in the presence of liquid nitrogen, then processed with the RNeasy midiprep Kit (Qiagen, Germany), following the manufacturer's protocol. Trace DNA contamination was removed from total RNA by a combination of acid phenol: chloroform 5:1 pH= 4.7 extraction and Dnase I treatment (Ambion, Texas). Real-time RT-PCR was performed on a Rotor-Gene RG-3000 (Corbett Research, Australia) using the Quantitect SYBR Green real-time RT-PCR kit (Qiagen, Germany), and the manufacturers protocols with 300 ng of Dnase I treated total RNA. Primers were designed to amplify a 108 bp fragment of the soybean  vspB  using the following primers: 5'-TGGTTCAACGCACTCTTC-3' and 5'-GGCTATGGTGAGCGTTCTTC-3'. Reverse transcription was performed for 30 min at 50°C followed by a 15 min denaturing at 95°C, and 40 cycles of 40 s at 95°C, 40 s at 58°C and 40 s at 72°C. Quantification was based on relative abundance to maize 18S RNA by amplifying a 174 bp fragment with primers: 5'-CCTGCGGCTTAATTGACTC-3' and 5'-GTTAGCAGGCTGAGGTCTCG-3', and using the comparative quantification function of the Rotor-Gene RG-3000 software. All real-time RT-PCR experiments were conducted in triplicate and on triplicate RNA preparations for each sample. Melting curve analysis and agarose gel electrophoresis were performed to verify single product formation.vspBWestern blot analysis Protein was extracted from 100 mg of leaves and stems with 0.5 ml of phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 ) supplemented with 1 tablet/10 ml buffer of the Complete-Mini protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN) by homogenizing in the presence of zirconia/silica 0.1 mm dia. beads. Centrifuged extract supernatants were removed and used for protein concentration determination [ 27 ]. Because of the low protein yields in stem extracts, they were precipitated with 10% trichloroacetic acid (TCA), washed with ice-cold acetone and resuspended prior to SDS-PAGE analysis. Thirty micrograms of protein were separated on 12% SDS-polyacrylamide gels. Proteins in the gel were either stained with silver nitrate [ 28 ] or transferred to Hybond-P (PVDF) membranes (Amersham Pharmacia Biotech, Inc. Piscataway, NJ) using Trans-Blot SD Semi-Dry Transfer Cell blotter and recommended protocols (Bio-Rad Laboratories). Soybean VSP was immunologically detected on the PDF membranes with anti VSPβ serum (provided by P. Staswick, University of Nebraska, produced as previously described [ 29 ] used at a 1:5,000 dilution. Detection was performed using a luminol substrate and the NEN Life Science Products, Inc. (Boston, MA) Reinascence kit. Chemiluminescent signal was captured by a Kodak Digital Science Image Station 440 Cf, and analyzed with Kodak 1D Scientific Image Software. Quantification of band intensity was always compared relative to samples from the same gel.
Western blot analysis
Protein was extracted from 100 mg of leaves and stems with 0.5 ml of phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 ) supplemented with 1 tablet/10 ml buffer of the Complete-Mini protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN) by homogenizing in the presence of zirconia/silica 0.1 mm dia. beads. Centrifuged extract supernatants were removed and used for protein concentration determination [ 27 ]. Because of the low protein yields in stem extracts, they were precipitated with 10% trichloroacetic acid (TCA), washed with ice-cold acetone and resuspended prior to SDS-PAGE analysis.Thirty micrograms of protein were separated on 12% SDS-polyacrylamide gels. Proteins in the gel were either stained with silver nitrate [ 28 ] or transferred to Hybond-P (PVDF) membranes (Amersham Pharmacia Biotech, Inc. Piscataway, NJ) using Trans-Blot SD Semi-Dry Transfer Cell blotter and recommended protocols (Bio-Rad Laboratories). Soybean VSP was immunologically detected on the PDF membranes with anti VSPβ serum (provided by P. Staswick, University of Nebraska, produced as previously described [ 29 ] used at a 1:5,000 dilution. Detection was performed using a luminol substrate and the NEN Life Science Products, Inc. (Boston, MA) Reinascence kit. Chemiluminescent signal was captured by a Kodak Digital Science Image Station 440 Cf, and analyzed with Kodak 1D Scientific Image Software. Quantification of band intensity was always compared relative to samples from the same gel.List of abbreviations RT-PCR, reverse transcriptase-polymerase chain reaction; VSP, vegetative storage protein
List of abbreviations
RT-PCR, reverse transcriptase-polymerase chain reaction; VSP, vegetative storage proteinAuthors' contributions MFG participated in experimental design, carried out the transgenic plant development, plant crosses, and molecular blotting/detection methods, and participated in manuscript draft preparation. RLS participated in experimental design, and provided guidance and training in development of transgenic maize. CM performed RT-PCR experiments. BTS participated in experimental design and provided expertise and training in plant crosses. RGS conceived of the study, participated in experimental design, coordinated the experimental plan, and wrote the draft manuscript. All authors read and approved the final manuscript.
Authors' contributions
MFG participated in experimental design, carried out the transgenic plant development, plant crosses, and molecular blotting/detection methods, and participated in manuscript draft preparation. RLS participated in experimental design, and provided guidance and training in development of transgenic maize. CM performed RT-PCR experiments. BTS participated in experimental design and provided expertise and training in plant crosses. RGS conceived of the study, participated in experimental design, coordinated the experimental plan, and wrote the draft manuscript. All authors read and approved the final manuscript.


In April 2002,  Science  published draft genome sequences for the two major subspecies of cultivated rice,  Oryza sativa . The release of the rice genome—the first plant crop to be sequenced—was big news. Rice is a staple crop for more than half of the world's population, and it was hoped that the availability of the genome sequence might enable scientists to develop more productive rice strains or strains that are more environmentally friendly. Furthermore, the rice genome might provide the key to understanding the genetics of other major cereal crops, all of which have much larger genomes.ScienceBut the sequences published in 2002 were only draft genomes, containing many gaps and errors—works-in-progress rather than finished products. Now, a large group of scientists led by the Beijing Institute of Genomics is publishing a much improved, near-complete genome analysis of the  indica  and  japonica  subspecies of  O. sativa , which are eaten in India and China, and Japan, respectively. Their analysis team, led by Gane Ka-Shu Wong, provides important insights into the evolution of rice.indicajaponicaFirst of all, the team improved their original whole-genome shotgun sequencing of  indica  by generating significantly more sequence data, and then they used better methods to assemble these data. In whole-genome shotgun sequencing, the entire genome is chopped into random fragments, each fragment is sequenced, and then powerful computer programs search for overlaps and put all these data in order. It's like putting a fiendishly difficult jigsaw puzzle together by looking for patches of matching color.indicaThe key to the improvement in the genome sequence analysis is that the researchers have used the combined DNA sequence data from the two subspecies to facilitate the sequence assembly. The result is a nearly 1,000-fold increase in contiguity for the two genome sequences. In other words, while the original draft was very fragmented, in the new version, 97.7% of the genes can be found, in either the  indica  or the  japonica  dataset, on one piece of DNA whose position along the chromosomes is well defined.indicajaponicaThe researchers have also used their improved genome sequence to investigate the evolutionary history of rice. Central to evolution is the development of new functions through mutation of existing genes. But when mutations occur in functional genes, the result is rarely beneficial, so it is thought that evolution is more likely to proceed first by duplicating existing genes and then experimenting on the “backup” copy of the gene. Wong and colleagues report that there is evidence in the rice DNA sequences for a whole-genome duplication event just before the grasses diverged from other flowering plants, about 55–70 million years ago. This genome duplication may have played a role in the origin of the grasses, which then spread rapidly across the world to provide important sources of food that, among other things, possibly influenced human evolution.Analysis of the rice genomes also indicates that a small chromosomal segment was duplicated about 21 million years ago and that there is massive ongoing duplication of individual genes. These individual gene duplications provide a continuous source of raw material for gene genesis and very likely contribute to the differences between members of the grass family. Now the challenge is to use the rice sequences as a basis for detailed genetic analyses of additional cereal crops and for the development of improved strains of not only rice, but wheat, maize, and other important food crops.