Myeong-Je Cho

A strategy for the identification of proteins targeted by thioredoxin

Thioredoxins are 12-kDa proteins functional in the regulation of cellular processes throughout the animal, plant, and microbial kingdoms. Growing evidence with seeds suggests that an h-type of thioredoxin, reduced by NADPH via NADP-thioredoxin reductase, reduces disulfide bonds of target proteins and thereby acts as a wakeup call in germination. A better understanding of the role of thioredoxin in seeds as well as other systems could be achieved if more were known about the target proteins. To this end, we have devised a strategy for the comprehensive identification of proteins targeted by thioredoxin. Tissue extracts incubated with reduced thioredoxin are treated with a fluorescent probe (monobromobimane) to label sulfhydryl groups. The newly labeled proteins are isolated by conventional two-dimensional electrophoresis: (i) nonreducing/reducing or (ii) isoelectric focusing/reducing SDS/PAGE. The isolated proteins are identified by amino acid sequencing. Each electrophoresis system offers an advantage: the first method reveals the specificity of thioredoxin in the reduction of intramolecular vs. intermolecular disulfide bonds, whereas the second method improves the separation of the labeled proteins. By application of both methods to peanut seed extracts, we isolated at least 20 thioredoxin targets and identified 5—three allergens (Ara h2, Ara h3, and Ara h6) and two proteins not known to occur in peanut (desiccation-related and seed maturation protein). These findings open the door to the identification of proteins targeted by thioredoxin in a wide range of systems, thereby enhancing our understanding of its function and extending its technological and medical applications.

Transformation of recalcitrant barley cultivars through improvement of regenerability and decreased albinism

Abstract During selection for transformed tissue, in vitro-cultured barley material rapidly loses regenerability or gives rise to albino plants, and this has caused difficulty in developing successful transformation technologies for important North American barley cultivars. Callus from three spring cultivars, Golden Promise (GP), Galena (GL), and Harrington (HT), was initiated from immature scutellar tissue and grown on callus-induction medium containing 2.5 mg/l of the auxin, 2,4-dichlorophenoxyacetic acid (2,4- d ), 0.01 or 0.1 mg/l of the cytokinin, 6-benzylaminopurine (BAP), and 5.0 μM cupric sulfate. The addition of BAP and copper, compared to auxin alone, resulted in shinier, more compact and slightly brown-colored callus, which was more regenerable. When the highly regenerable structures were exposed to dim light and maintained on 0.1 mg/l BAP, they could be cultured for more than a year without a marked loss in regenerability or evidence of albinism. When GP tissues were initiated on auxin alone (2,4- d or dicamba) and transferred to 2,4- d , BAP and copper, as an intermediate step before regeneration, green shoot production increased 2.4 to 11.4 times for both transgenic and nontransgenic calli. Similar increases were found for nontransgenic GL and HT. This increase in regenerability, likely due to a change in the developmental state of the cultures, along with other changes in the transformation protocol, resulted in successful transformation of the previously recalcitrant GL and HT cultivars.

Transgenic barley grain overexpressing thioredoxin shows evidence that the starchy endosperm communicates with the embryo and the aleurone

Homozygous lines of barley overexpressing a wheat thioredoxin h transgene (up to 30-fold) were generated earlier by using a B1-hordein promoter with a signal peptide sequence for targeting to the protein body and found to be enriched in starch debranching enzyme (pullulanase). Here, we describe the effect of biochemically active, overexpressed thioredoxin h on germination and the onset of α-amylase activity. Relative to null segregant controls lacking the transgene, homozygotes overexpressing thioredoxin h effected (i) an acceleration in the rate of germination and appearance of α-amylase activity with a 1.6- to 2.8-fold increase in gibberellin A1 (GA1) content; (ii) a similar acceleration in the appearance of the α-amylase activity in deembryonated transgenic grain incubated with gibberellic acid; (iii) a 35% increase in the ratio of relative reduction (abundance of SH) of the propanol soluble proteins (hordein I fraction); and (iv) an increase in extractable and soluble protein of 5–12% and 11–35%, respectively. Thioredoxin h, which was highly reduced in the dry grain, was degraded in both the null segregant and homozygote after imbibition. The increase in α-amylase activity and protein reduction status was accompanied by a shift in the distribution of protein from the insoluble to the soluble fraction. The results provide evidence that thioredoxin h of the starchy endosperm communicates with adjoining tissues, thereby regulating their activities, notably by accelerating germination of the embryo and the appearance of α-amylase released by the aleurone.

Production of transgenic tall fescue and red fescue plants by particle bombardment of mature seed-derived highly regenerative tissues

Abstract Highly regenerative tissues of tall fescue and red fescue produced from mature seed-derived embryogenic callus were induced and proliferated on medium containing 2,4-dichlorophenoxyacetic acid (4.5 or 9.0 μM), 6-benzylaminopurine (0, 0.044, 0.44 or 2.2 μM) and cupric sulfate (0.1 or 5.0 μM) under dim-light conditions (10 to 30 μE m–2 s–1, 16 h light). Tall fescue tissues were transformed with three plasmids containing the genes for hygromycin phosphotransferase (hpt), phosphinothricin acetyltransferase (bar) and β-glucuronidase (uidA;gus), and red fescue with three plasmids containing hpt, uidA and a synthetic green fluorescent protein gene [sgfp(S65T)]. DNA from T0 plants of eight independently transformed lines from tall fescue and 11 from red fescue were analyzed by PCR and DNA blot hybridization. The co-expression frequency of all three transgenes [hpt/bar/uidA or hpt/uidA/sgfp(S65T)] in transgenic tall fescue and red fescue plants was 25–27%; for two transgenes [hpt/bar or hpt/uidA for tall fescue and hpt/uidA or hpt/sgfp(S65T) for red fescue], the co-expression frequency was 50–75%.

Inheritance of tissue-specific expression of barley hordein promoter- uidA fusions in transgenic barley plants

Abstract Barley (Hordeum vulgare L.) hordeins are alcohol-soluble redundant storage proteins that accumulate in protein bodies of the starchy endosperm during seed development. Strong endosperm-specific β-glucuronidase gene-(uidA; gus) expression driven by B1- and D-hordein promoters was observed in stably transformed barley plants co-transformed with the selectable herbicide resistance gene, bar. PCR analysis using DNA from calli of 22 different lines transformed with B1- or D-hordein promoter-uidA fusions showed the expected 1.8-kb uidA fragment after PCR amplification. DNA-blot analysis of genomic DNA from T0 leaf tissue of 13 lines showed that 12 (11 independent) lines produced uidA fragments and that one line was uidA-negative. T1 progeny from 6 out of 12 independent regenerable transgenic lines tested for uidA expression showed a 3 : 1 segregation pattern. Of the remaining six transgenic lines, one showed a segregation ratio of 15 : 1 for GUS, one expressed bar alone, one lacked transmission of either gene to T1 progeny, and three were sterile. Stable GUS expression driven by the hordein promoters was observed in T5 progeny in one line, T4 progeny in one line, T3 progeny in three lines and T2 or T1 progeny in the remaining two fertile lines tested; homozygous transgenic plants were obtained from three lines. In the homozygous lines the expression of the GUS protein, driven by either the B1- or D-hordein promoters, was highly expressed in endosperm at early to mid-maturation stages. Expression of bar driven by the maize ubiquitin promoter was also stably transmitted to T1 progeny in seven out of eight lines tested. However, in most lines PAT expression driven by the maize ubiquitin promoter was gradually lost in T2 or later generations; one homozygous line was obtained. In contrast, six out of seven lines stably expressed GUS driven by the hordein promoters in T2 or later generations. We conclude that the B1- and D-hordein promoters can be used to engineer, and subsequently study, stable endosperm-specific gene expression in barley and potentially to modify barley seeds through genetic engineering.

High-frequency transformation of oat via microprojectile bombardment of seed-derived highly regenerative cultures

A highly efficient and reproducible transformation system for oat (A6ena sati6a L. cv. GAF:Park-1) was developed using microprojectile bombardment of highly regenerative tissues derived from mature seeds. Callus was induced under dim light conditions on medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and high cupric sulfate. Highly regenerative tissues, generated from embryogenic callus, were used as a transformation target. From 327 individual explants bombarded with the b-glucuronidase gene (uidA; gus) and a hygromycin phosphotransferase gene (hpt), 84 independent transgenic events were obtained after an 8‐12-week selection period on hygromycin. All events were regenerable, giving an effective transformation frequency of 26%; co-expression of GUS activity occurred in 70% of the independent events. Presence of the foreign genes in DNA from leaf samples of T0 and T1 plants was confirmed by polymerase chain reaction (PCR) amplification and:or DNA blot hybridization. Fertility of the plants from the transgenic lines was 63% (24:38) and the transgene(s) was stably transmitted to T1 and T2 progeny.

The Level of Expression of Thioredoxin is Linked to Fundamental Properties and Applications of Wheat Seeds

Work with cereals (barley and wheat) and a legume (Medicago truncatula) has established thioredoxin h (Trx h) as a central regulatory protein of seeds. Trx h acts by reducing disulfide (S-S) groups of diverse seed proteins (storage proteins, enzymes, and enzyme inhibitors), thereby facilitating germination. Early in vitro protein studies were complemented with experiments in which barley seeds with Trx h overexpressed in the endosperm showed accelerated germination and early or enhanced expression of associated enzymes (alpha-amylase and pullulanase). The current study extends the transgenic work to wheat. Two approaches were followed to alter the expression of Trx h genes in the endosperm: (1) a hordein promoter and its protein body targeting sequence led to overexpression of Trx h5, and (2) an antisense construct of Trx h9 resulted in cytosolic underexpression of that gene (Arabidopsis designation). Underexpression of Trx h9 led to effects opposite to those observed for overexpression Trx h5 in barley-retardation of germination and delayed or reduced expression of associated enzymes. Similar enzyme changes were observed in developing seeds. The wheat lines with underexpressed Trx showed delayed preharvest sprouting when grown in the greenhouse or field without a decrease in final yield. Wheat with overexpressed Trx h5 showed changes commensurate with earlier in vitro work: increased solubility of disulfide proteins and lower allergenicity of the gliadin fraction. The results are further evidence that the level of Trx h in cereal endosperm determines fundamental properties as well as potential applications of the seed.

Selection and osmotic treatment exacerbate cytological aberrations in transformed barley (Hordeum vulgare)

Summary A much higher percentage (78 percnt;) of karyotypically abnormal cells were observed in transgenic barley callus cultures, compared with the 15 percnt; cytological abnormality in cells from nontransgenic callus cultures. Out of 22 independently transformed callus lines generated by microprojectile bombardment of immature embryos, only 7 lines had a relatively high percentage (35-76 percnt;) of diploid chromosomes (2n=2x=14); 5 (71 percnt;) of these lines were regenerable. Of the remaining 15 lines a high percentage (92-100 percnt;) of cells in these lines had cytological abnormalities, primarily ploidy changes [tetraploidy (2n=4x=28), octaploidy (2n=8x=56) and aneuploidy] and structural variations; 8 (53 percnt;) of these lines were regenerable. There was a high correlation between the cytological status of callus tissues and their regenerated plants. We studied which factor(s) in the transformation process were responsible for the increased cytological aberrations. Only 18-21 percnt; of 6- and 12-week-old callus cells had observable cytological changes in the absence of transformation. In contrast, imposing osmotic or selection treatment triggered extensive cytological aberrations, 41 percnt; and 40-46 percnt; in 6-week-old callus cultures and 60 percnt; and 48-51 percnt; in 12-week-old callus cultures, respectively. Bombardment itself did not appear to affect the frequency of cytological aberration in cells of callus cultures.

Transgenic Cereals: Hordeum vulgare L. (barley)

The development of barley as a crop dates to the earliest agricultural activities of humans, and it remains one of the major cereals grown for feed and food, and for the production of beer. In this century, an understanding and application of quantitative genetic theory has created a genetically elite crop that is divergent from its ancestors. Further improvements in barley cultivare will depend on continued access to useful allelic variability. Sexual hybridization will continue to play an important role in such improvement, but its utility is limited because potentially useful alleles are either linked to undesirable alleles or unavailable because of sexual incompatibilty. The advent of molecular genetics and nonsexual gene transfer offers exciting opportunities to bypass these limitations and to provide access to more diverse sources of genes. Recent developments have added barley to the list of major crops that are amenable to this type of genetic manipulation either through direct DNA transfer (bombardment) or mediated by Agrobacterium tumefaciens. However, significant problems remain, and include: 1) the lack of reproducible, efficient transformation systems for commercial germplasm; 2) the induction of stable genetic and epigenetic changes during the in vitro process; and 3) transgene and transgene expression instability. In this chapter, we will discuss and describe the first systems used for the genetic transformation of barley, introduce and describe the development of new systems for barley transformation, and comment on past and future uses of barley transformation as a tool for basic science and commercial application.

Stable transformation of a recalcitrant kentucky bluegrass (Poa pratensis L.) cultivar using mature seed-derived highly regenerative tissues

SummaryAn efficient method to produce highly regenerative tissues from seeds of a previously recalcitrant cultivar of Kentucky bluegrass (Poa pratensis L. ev. Kenblue) was established under dim-light conditions (10–30 μE m−2s−1, 16-h light) using media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5 or 9.0 μM), 6-benzylaminopurine (BA; 0.44 or 2.2 μM), and a high level of cupric sulfate (5.0 μM). The tissues were co-transformed with three plasmids containing the genes for hygromycin phosphotransferase (hpt), β-glucuronidase (uidA; gus), and a synthetic green fluorescent protein gene [sgfp(S65T)]. From 463 individual explants bombarded, 10 independent transgenic events (2.2%) were obtained after a 3–4-month selection period for hygromycin resistance using 30–100 mg l−1 hygromycin B; of the 10 independent events, seven (70%) were regenerable. Stable integration of the transgene(s) in transgenic plants was confirmed by polymerase chain reaction and DNA blot hybridization analyses. Coexpression frequency of all three genes was 20%; for two transgenes, either hpt/uidA or hpt/sgfp(S65T), coexpression frequency was 30–40%.