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Cultivar-Specific Seedling Vigor and Expression of a Putative Oxalate Oxidase Germin-Like Protein in Sugar Beet (Beta Vulgaris L.)

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Cultivar-Specific Seedling Vigor and Expression of a Putative Oxalate Oxidase Germin-Like Protein in Sugar Beet (Beta Vulgaris L.) Theor Appl Genet (2003) 107:54–61 DOI 10.1007/s00122-003-1229-9 Benildo G. de los Reyes · J. Mitchell McGrath Cultivar-specific seedling vigor and expression of a putative oxalate oxidase germin-like protein in sugar beet (Beta vulgaris L.) Received: 12 November 2002 / Accepted: 16 December 2002 / Published online: 21 March 2003 Springer-Verlag 2003 Abstract For genetic screening and breeding purposes, Introduction an in vitro germination system that reflects relative field emergence potential was used to screen for germination- Field emergence of sugar beet (Beta vulgaris) is an annual enhancing and stress-induced genes from germinating concern for growers, and improving emergence potential seedlings from two varieties of sugar beet. Three full- has not been amenable to selection due to developmental length germin-like protein (GLP) gene classes were and environmental influences on seed quality. On aver- recovered from stress-germinated seedlings of a superior age, 60% of the planted seed contributes to the maximum emerging variety. GLP gene expression, oxalate oxidase stand count in Michigan (Anonymous 1997), with a range protein activity, the H2O2 content of stressed seedlings, of emergence from 0 to 90% depending on environmental but not catalase activity, were induced by stress germi- and disease pressure during stand establishment. Field nation conditions (e.g. excess water, NaCl, mannitol, or emergence and stand establishment are largely deter- oxalate) in a good emerging hybrid and were not induced mined by seedling vigor, which has been defined as the in a poor emerging variety. Only one of the three germin- ability of physiologically viable seed to germinate and like protein genes (BvGer165) was differentially regulat- flourish in diverse environments (Kneebone 1976). For ed, and was induced only in the good emerger. Hydrogen sugar beet, the first 8 weeks of growth are most critical peroxide promoted germination and partially compensat- (Durr and Boiffin 1995). Improving emergence potential ed solute-depressed germination percentages. Unlike is problematic because both the seed germination envi- other solute recovery by hydrogen peroxide regimes, ronment the and seed production environment profoundly recovery in oxalic acid plus H2O2 was cultivar-indepen- influence seedling performance (Johnson and Burt 1990). dent. A block in oxalate metabolism is postulated to Germination is the first phase of emergence, resulting contribute to lower germination under stress in the lower from a culmination of events such as imbibition of water emerging variety. Selection for stress-induced germin that initiates metabolic activity and leads to subsequent expression, or for down-stream targets, presents the first cell enlargement and division (Thomas 1993; Copeland direct target to enable breeding for improved field and McDonald 1995). emergence of sugar beet. A novel stress test appeared promising for assigning relative field emergence potential, demonstrating that Keywords Germination · Abiotic stress · Hydrogen germination of commercial quality sugar beet seed (e.g. peroxide · Seedling vigor · Molecular phenotyping >92% germ) can be discriminated in vitro to reveal higher and lower vigor seedlots. Germination in water showed the same relative ranking of varieties as their emergence Communicated by H.F. Linskens in the field, and germination in hydrogen peroxide B. G. de los Reyes · J. M. McGrath ()) solution promoted germination to the extent observed USDA-ARS, Sugar Beet and Bean Research Unit, with traditional germination methods used in commercial 494 PSSB, Michigan State University, quality assessment (McGrath et al. 2000). In this report, East Lansing, MI 48824-1325, USA we have begun the search for possible reasons of e-mail: [email protected] enhanced in vitro germination of sugar beet by hydrogen Tel.: (517)-432-2355 Fax: (517)-337-6782 peroxide, since the effect is dramatic, and has potential in breeding for enhanced emergence. B. G. de los Reyes We examined two commercially acceptable sugar beet University of Arkansas, 115 Plant Science Building, hybrids known to differ in relative field emergence. We Dept of Crop, Soil, and Environmental Sciences, show that the good emerging commercial hybrid specif- Fayetteville, AR 72701, USA 55 ically induced expression of a gene with high similarity to RNA gel-blot analyses known germin-like protein (GLP) genes, and that this Eight hundred nanograms of polyA+ mRNA were fractionated in gene is not induced in the lesser emerging commercial 1.2% formaldehyde-agarose gels, blotted on nylon membranes hybrid. Expression of this particular germin-like protein (HybondN, Amersham) and hybridized with 32P-dCTP-labeled appears to be a physiological marker for enhanced sugar BvGer171 overnight at 42 C in NorthernMax Prehyb/Hyb buffer beet germination and, by extension, field emergence. We (Ambion). Post-hybridization washes (twice, 15 min each) in 2 SSC, 0.1% SDS followed with 0.5 SSC, 0.1% SDS preceded propose that the specific GLP induced is an oxalate exposure to X-ray film (3 days). Filters were stripped and re- oxidase based on correlative evidence of increases in hybridized with an actin probe (as a control) following the same oxalate oxidase activity and internal hydrogen peroxide procedure. The actin clone was obtained from the sugar beet EST concentrations, and suggest that the oxalate oxidase collection (Genbank accession no. AWO63023). metabolic product, hydrogen peroxide, markedly increas- es expression of stress-related genes during aqueous Reverse transcription-PCR (RT-PCR) germination regimes and leads to enhanced seedling vigor. Total RNA (1 mg) from each of USH20 and ACH185 filter-paper and solution-germinated seedlings were reverse transcribed using RT-for-PCR (Clontech) as per manufacturer’s instructions. Specific primer pairs to each full-length recovered germin cDNA were Materials and methods designed, synthesized and used to amplify their cognate cDNAs from 1 mg of total RNA using dNTP mix (50 mM), 33P-dATP Seed germination (50 nM, 2 mCi) and Advantage KlenTaq polymerase mix (2 units) (Clontech). Gene specific primers were designed from accession The same seedlots of two commercial sugar beet (Beta vulgaris L. nos. AF10016 (BvGer165), AF10017 (BvGer171) and AF10018 ssp. vulgaris var. altissima Dll.) hybrids, USH20, a diploid (Coe (BvGer172) as shown: and Hogaboam 1971), and ACH185, a triploid (American Crystal, – BvGer165: Forward: 5'-TGTACCCGGAAACAATGTA; Re- Moorhead, Minn.), with commercially acceptable germination verse: 5'-TGTTGTCATCATACACT percentages (>92%) were used throughout these experiments. – BvGer171: Forward: 5'-GTTTCCCTTAGCTTGCAAAGTA; Standard germination assays were performed on moist pleated Reverse: 5'-CATTTGCACAAACTTCCAAAG germination paper (TeKrony and Hardin 1966; Bornscheuer et al. – BvGer172: Forward: 5'-GTTTCCCTTAGCTTGCAAAAATA; 1993). Germination in solution was performed as described Reverse: 5'-TATCATTCAATACTGCAAAGGA. (McGrath et al. 2000). Briefly, four replicates of 25 seeds each PCR cycling was 1 min at 95 C, 22 cycles of 30 s at 94 C, were immersed in 15 ml of solution, and germination counts were 1 min at 50 C, 1 min at 72 C; and final extension for 10 min at done 96 h post-immersion, with an intermediate count and change 72 C. Fragments of 515 bp, 831 bp and 850 bp, amplified with of solution at 48 h. A seed was scored as germinated when the gene specific primers to BvGer165, BvGer171 and BvGer172, radical protruded from the seed ball. All values reported are respectively, were verified by sequencing. Actin at (675-bp germination percentages after the total 96-h incubation. Germina- fragment amplified with primers Forward = 5'-TGTGAGCAA- tion treatments in solution included 150 mM of NaCl, 200 mM of CTGGGATGA and Reverse = 5'-GGTAACATCGTGCTCAGT) mannitol, 18 MOhm of water, 88 mM of H2O2, 120 mM of oxalic was used as a control for RT-PCR to verify amplification and acid pH 4.0 and some combinations of these. Field emergence was loading equivalency. Amplification products were separated in 6% assessed using six replicates of 100 seeds/10 m plot in each of 3 non-denaturing polyacrylamide, dried, and autoradiographed over- years at the Saginaw Valley Bean and Beet Research Farm night. The specificity of each primer pair was confirmed by the lack (Saginaw, Mich.). of cross-amplification with non-target BvGer plasmid clones, and by re-sequencing the products amplified from experimental sam- ples. Differential display RNA was isolated from 1,000 to 1,500 4-day old seedlings Oxalate oxidase assay (Logeman et al. 1996). First-strand cDNAs were synthesized from 1 mg of total RNA (pre-treated with DNAse I) with oligo-dT primer Crude extracts of oxalate oxidase were prepared from 4-day old (1 mM) and MMLV reverse transcriptase (1,000 units) at 42 C for filter paper, and solution-germinated USH20 and ACH185 seed- 1 h. Differential display (DD-PCR) was performed on USH20 total lings, via the method of Pundir and Nath (1984) with slight RNA samples using the Delta Differential Display kit (Clontech) modifications. Germinated seedlings (15 g) were excised from the following the manufacturer’s instructions. Amplified products were ruptured pericarp and homogenized in 15 ml of distilled water at fractionated in 6% denaturing polyacrylamide gel (Sambrook et al. 4 C. The homogenate was squeezed through a double layer of 1989), dried, and autoradiographed for 24 h. Fragments with signal cheesecloth and centrifuged at 4 C, 15,000 g for 40 min. differences between control (filter paper germinated seedlings) and (NH4)2SO4 crystals were added to the supernatant to obtain 0– treatments were excised, re-amplified, cloned in pTAdv TA vector 30% and 30–65% saturation fractions, and crude protein extracts (Clontech) and sequenced. A germin-like DD-PCR fragment precipitated at 4 C overnight. The precipitate from 30 to 65% (amplified with the P4/P4 primer combination) was used to recover fractions was collected by centrifugation at 4 C 10,000 g for 14 full-length cDNA clones from a Lambda Zap cDNA library 40 min, and the pellet was re-dissolved in 0.05 M sodium succinate (Stratagene) constructed from USH20 4-day old solution-germi- pH 5.0 and stored at 4 C.
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