Executive Summary

This report represents the collaborative research by the laboratories of Dr. Henry Nguyen (Missouri) and Hans Bohnert (Illinois) with a focus on soybean transformation with gene constructs that are intended to increase tolerance of the plants to oxidative stresses, drought and low temperature. In the first year we have transformed soybean plants with the ipt gene construct, for localized cytokinin production and the molecular analysis for the confirmation of transgene integration were completed. The T0 and T1 generation seeds were collected for further analysis. Transformation events with trehalose biosynthetic fusion gene construct is in progress. The initial part of the project, for which gene constructs exist, will express genes for the synthesis of ectoine in soybeans. This work has met with unexpected difficulties and is not completed. Another strategy involves the expression of a transcription factor in constitutive and stress-inducible fashion. We made gene constructs with arabidopsis CBF3 and CBF4 transcription factors for soybean transformation to understand the signaling regulatory network. Also, we suggested focusing future strategies of crop improvement on the expression of genes that are in a hierarchical way above the transcription factor level. Unfortunately, the lack funding affected further experiments and progress with these aspects.

Research progress and preliminary results

1. Expression of isopentenyl transferase gene

In plants, senescence is accompanied by changes in endogenous ethylene, abscisic acid (ABA), and cytokinins, and these changes are believed to mediate signaling events that control the process. Several laboratories have generated transgenic plants with enhanced cytokinin levels by transformation with the isopentenyl transferase (ipt) gene cloned from Agrobacterium tumefaciens (Heidekamp et al 1983). We have generated number of transgenic soybean plants with ipt gene (isopentenyl transferase) under the control of senescence specific SAG12 promoter. The ipt gene encodes the enzyme isopentenyl transferase which catalyses the initial rate limiting step in cytokinin biosynthesis where isopentenyl pyrophosphate is condensed with adenosine monophosphate to produce isopentenyl AMP (Akiyoshi et al, 1984; Barry et al, 1984). The promoter region of a senescence-associated gene from Arabidopsis, SAG12 drives the tissue specific expression of ipt gene in leaves and delay senescence (Gan and Amasino, 1995). Various labs have reported the delayed leaf senescence in SAG12-IPT transformed plants (Zhang et al., 2000; McCabe et al., 2001). Sustained cytokinin content also enhances flooding and drought tolerance (Dervinis, 1999; Zhang et al., 2000).

For our transformation experiments entire region of the senescence promoter, the ipt coding region and the NOS termination regions were excised from pSG516 and finally cloned in the soybean transformation vector pZY101 carrying bar as plant selectable marker.

Fig 1. Ipt gene construct.

Primary (T0) transgenic soybean lines were developed using genotype Williams 82 and our improved Agrobacterium-mediated transformation of soybean cotyledonary-node system. All T0 putatively transgenic plants were subjected to the leaf painting and molecular analysis to confirm the integration of the transgene. T1 generation evaluation is in progress.

Fig 2. Generation of soybean transgenic plants with SAG12-ipt gene construct. (a) Regeneration from soybean cotyledonary-node explants. (b) Mature transgenic soybean plant (T0)bearing pods. (c) T1 generation plant.

2. Soybean transformation with Trehalose phosphate synthase fusion gene

Trehalose is a non-reducing disaccharide of glucose functions as a compatible solute in the stabilization of biological structures under abiotic stress (Garg et al., 2002). Overexpression of the E. coli trehalose biosynthetic genes (ostA and ostB encoding TPS and TPP) as a fusion gene help accumulate trehalose and protect the soybean plants from the oxidative and drought stress damages. We transform the soybean with the TPSP fusion gene under the control of the novel stress inducible promoter ABRC. The Agrobacterium LBA4404 is transformed with the binary vector carrying ABRC promoter, TPSP, and pin terminator and the Soybean transformation events are in progress.

Fig 3. TPSP gene construct with stress inducible promoter.

3. Soybean transformation with CBF3 and CBF4 transcription factors

Haake et al (2002) isolated an apparent homolog of the CBF/DREB1 proteins (CBF4) that plays the equivalent role during drought adaptation. In contrast to the three already identified CBF/DREB1 homologs, which are induced under cold stress, CBF4 gene expression is up-regulated by drought stress, but not by low temperature. CBF4 over-expression in transgenic Arabidopsis plants results in the activation of C-repeat/dehydration-responsive element containing downstream genes that are involved in cold acclimation and drought adaptation.

Arabidopsis cbf3 and cbf4 were PCR cloned with specific restriction sites and used for the further subcloning processes. Gene constructs were made with ABRC promoter and 3' vsp terminator and mobilized to the soybean transformation binary vector pZY101. The agrobacterium mediated transformation of soybean with the cbf3 and cbf4 under the control of stress inducible promoter is in progress.

4. Update on Ectoine Biosynthesis and Function:

The vectors for ectoine biosynthesis under inducible promoters in soybean (replacing constitutive promoters) have not yet been completed. However, with all three genes expressed in tobacco, we have been able to carry out some additional analyses.

Analyses with transgenic tobacco plants generated the following data points. Additional work would be required to clean up the materials. However, a Japanese group has taken information that we presented at a conference and has advanced the work (Nakayama et al., 2000). Rontein et al. (2002) have discussed ectoine as an osmoprotectant. Also, it should be pointed out that ectoine does indeed act in such a role in a number of bacterial species and it appears to also act as UV-protectant (Bunger and Driller, 2004; Onraedt et al., 2004; Jebbar et al., 2005).

• EctB overexpression alone leads to death, most likely due to GABA accumulation, GABA has recently been suggested to constitute a signaling molecule (Fait et al., 2005).
• Tobacco lines maintained and analyzed (11) in detail indicated that the 3 genes are expressed in all lines.
• Only small amounts of ectoine were detected by MS/NMR - ~ 10-250 nM.
• Surprisingly, all transgenovars contain elevated potassium (x 2-3), glutamine/glutamate, aspartate, arginine and very large amounts of proline (x 5 - 50)
• germination on up to 250 mM NaCl indicated protection although no high accumulation of ectoine was detected.
• Withholding water from mature plants for nearly four weeks eliminated all SR1 control plants, while the ECT+ plants lost all developed leaves, but their meristems remained turgescent and, after watering, developed new leaves.

References

Akiyoshi DE, Klee H, Amasino RM, Nester EW, Gordon NW (1984) T-DNA of Agrobacterium tumefaciens encodes an enzyme of cytokinin biosynthesis. Proc Natl Acad Sci USA 81:5994-5998.

Barry GF, Rogers SG, Fraley RT, Brand L (1984) Identification of a cloned cytokinin biosynthetic gene. Proc Natl Acad Sci USA 81:4776-4780

Buenger J, Driller H (2004). Ectoin: an effective natural substance to prevent UVA-induced premature photoaging. Skin Pharmacol Physiol. 17: 232-7.

Fait A, Yellin A, Fromm H (2005). GABA shunt deficiencies and accumulation of reactive oxygen intermediates: insight from Arabidopsis mutants. FEBS Lett. 579: 415-20

Gan S, Amasino RM (1995) Inhibition of leaf senescence by autoregulated production of cytokinin. Science 270: 1986-1988

Garg A, Kim J, Owens TG, Ranwala AP, Cho YD, Kochian LV and Wu RJ (2002) Trehaloseaccumulation in rice plant confers high tolerance levels to different abiotic stresses. Proc Natl Acad Sci USA 99:15898-15903.

Haake V, Cook D, Riechmann JL, Pineda O, Thomashow MF and Zhang JZ (2002) Transcription Factor CBF4 Is a Regulator of Drought Adaptation in Arabidopsis. Plant Physiol 130:639-648.

Heidekamp F, Dirkse WG, Hille J, van Ormondt H. (1983) Nucleotide sequence of the Agrobacterium tumefaciens octapine Ti plasmid-encoded tmr gene. Nucl Acids Res 11: 6211-6233

Jebbar M, Sohn-Bosser L, Bremer E, Bernard T, Blanco C (2005). Ectoine-induced proteins in Sinorhizobium meliloti include an Ectoine ABC-type transporter involved in osmoprotection and ectoine catabolism. J Bacteriol. 187: 1293-304

Nakayama H, Yoshida K, Ono H, Murooka Y, Shinmyo A (2000). Ectoine, the compatible solute of Halomonas elongata, confers hyperosmotic tolerance in cultured tobacco cells.
Plant Physiol. 122: 1239-47

Onraedt A, De Muynck C, Walcarius B, Soetaert W, Vandamme E (2004). Ectoine accumulation in Brevibacterium epidermis. Biotechnol Lett. 26: 1481-5.

Rontein D, Basset G, Hanson AD (2002). Metabolic engineering of osmoprotectant accumulation in plants. Metab Eng. 4: 49-56.