Developmental changes in abundance of the VSPbeta protein following nuclear transformation of maize with the soybean vspbeta cDNA

Abstract

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.

Results: From 81 bombardments, 101 plants were regenerated, and plants from five independent lines produced vspB transcripts and VSPbeta polypeptides. In leaves from seven-week-old plants (prior to flowering), VSPbeta accumulated to 0.5% of the soluble leaf protein in primary transgenic plants (R0), but to only 0.03% in R1 plants. During seed-filling (silage-stage) in R1 plants, the VSPbeta 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 VSPbeta protein became unstable during seed-fill.

Conclusion: Developmental differences in the accumulation of soybean VSPbeta when transgenically expressed in maize show that despite no changes in the vspB transcript level, VSPbeta protein that is readily detected in leaves of preflowering plants, becomes undetectable as seeds begin to develop.

Figure 1

Southern blot analysis of primary (R₀) maize transformed with pAHC25 and pRSVP-1. Twenty μgs of EcoRI restricted genomics DNA were electrophoresed through 0.8% agarose and blotted to Hybond membrane. The membranes were probed with either digoxigenin labeled bar (a) or vspB (b). Numbers and lines on sides of blots indicate location of molecular size markers (the number represents size in kilobases). The "a" and "b" sections are aligned so that the same genomic DNA samples are vertically aligned and represented by the same lane label. Plasmid lane is the plasmid containing the either the bar or the vspβ clones used as a positive control. Untransformed controls are lanes containing genomic DNA from untransformed Hi-II maize. Soybean indicates lanes containing restricted soybean genomic DNA. The two panels within each section represent separate blots hybridized with the same probe.

Figure 2

Western blot detection of VSPβ in primary (R₀) transgenic maize. Thirty micrograms of protein from each sample were separated by SDS-PAGE. (a) Silver-stained 12% SDS-PAGE polypeptide profile for six of the 14 analyzed R₀ plants. (b) Immunodetection of VSP protein in SDS-PAGE separated transgenic R₀ maize extracts transferred to Hybond-P membranes and immunodetected using VSPβ antiserum and the Reinascence kit chemiluminescent detection method (NEN Life Sciences Products, Inc). (c) Underexposed western blot showing two distinct bands corresponding to VSPα and VSPβ polypeptides in soybean leaves.

Figure 3

Southern blot detection of vspB in R₁ transgenic maize. Twenty micrograms of EcoRI restricted genomics DNA was electrophoresed through 0.8% agarose and blotted to Hybond membrane. The membrane was probed with digoxigenin labeled vspB. The alphabetical labels of each R₁ family represent individual R₁ plants. Control sample represent genomic DNA from untransformed maize Hi-II. Two positive control samples are also included: plasmid, pRSVP-1 restriction digested to liberate the vspB cDNA clone (only partial digestion occurred as evidenced by the two bands at higher molecular weight than the vspB fragment), and genomic DNA from the 45-1-2 R₀ plant, previously shown to contain the vspB sequence. The Eco RI restricted soybean genomic DNA is also included as a positive control for hybridization of the soybean vspB probe.

Figure 4

(a) Northern blot detection of vspB transcripts in R₁ transgenic maize. Thirty micrograms of total RNA were separated on 1.2% agarose formaldehyde gels and blotted to Hybond N⁺ membranes. The vspB transcripts were detected by hybridized with a digoxigenin labeled vspB probe. (b) Western blot detection of VSP in R₀ and their progeny (R₁) transgenic maize. Thirty micrograms of protein in extracts from leaves of 7 weeks old plants were separated on 12% SDS-PAGE, blotted onto Hybond-P membrane, and VSP was immunodetected using VSPβ antiserum and the Renascence kit chemiluminescent detection method (NEN Life Sciences Products, Inc).

Figure 5

a) Real-time RT-PCR quantification of vspB transcripts in R₁ transgenic maize tissue. Two hundred micrograms of total RNA from the indicated tissue was used as the template source for real-time RT-PCR detection of vspB transcripts. Reactions were performed in 15 uL volume using the Qiagen Quantitect SYBR Green kit. Expression values are calculated by normalizing all threshold cycles (C_t) for vspB to the 18S rRNA C_t and converting this value to fold-increase over the value for the lowest expressing tissue, *44-1-1-stem, which was arbitrarily set at 1.0. b,c) Developmental changes in VSPβ level in transgenic maize vegetative tissues. Soybean VSPβ was immunodetected in Western blots of 30 μgs of total protein from crude extracts separated by 12% SDS-PAGE and blotted to Hybond-P membranes. Crude Extracts were prepared from: YL, leaves from preflowering plants; SL, leaves from silage stage plants; and SS, stems from silage stage plants. (b) Coomassie blue stained SDS-PAGE separated proteins from crude extracts; (c) VSP was immunodetected using VSPβ antiserum and the Renascence kit chemiluminescent detection method (NEN Life Sciences Products, Inc). The arrows indicate the VSP-β protein band.