Development of Stress Tolerant Soybean through SIZ1 Gene Overexpression (In Progress)

Clara Richardson, Nathan Boehm, Lynda McMaster-Schuyler and Peiyu ZengBiotechnology Program, Department of Natural Sciences and Mathematics, College of Agriculture and Technology, State University of New York, Cobleskill, NY 12043

Abstract

A variety of traits have a relationship to yield and can improve the overall soybean (Glycine max) plant by manipulating the expression of the nucleotide encoding SIZ1 polypeptide.

SUMO modification, specifically the regulation of SUMO E3, a small plant modifier, has been discovered to control the regulation of immunity, cell cycle progression including mitosis, DNA repair and chromatin stability. The focus of the sumoylation within plants is involved

with the response of abiotic and biotic stresses as well as the overall development of the plant and flower. By modulating the SIZ1 polypeptide the plant responds with increased yield [2].

SIZ1 is required for the sumoylation of the cytoskeleton proteins and the replication of PCNA, a factor of sumoylation. The overexpression of the SIZ1 gene can result in increased growth and photosynthetic activity. The additional expression can also increase the plants ability to adapt to stress factors. These factors include adaptability to increased drought conditions,

temperature fluctuations and nutrient deprivation of phosphate [4]. 

Keywords: SUMO Modification, Sumoylation, SIZ1 Gene

Background

Soybean is one of the most widespread commodities that accounts for a large contribution to the world food supply. This economically valued crop provides protein, oil, fiber, bio-diesel and livestock grain. The production of this crop faces challenges that affect the overall health of the plant as well as overall yields.

Plant transformation through Agrobacterium-mediated means allows the generation of cultivars that are genetically resistant to

stress factors and therefore pose a greater ability to overcome sub-optimal conditions. The overexpression of the SIZ1 gene has demonstrated an increase in photosynthesis activity as well as an

improved response to an increase in temperature and drought. The current study focuses on the development of soybean with an

additional SIZ1 gene expressed. The ability to genetically engineer an additional copy of this gene could generate a cultivar that has increased yields due to a greater adaptability to growing

conditions that cause stress [4].

Research Methods

Sterile Glycine max cultivar Williams 82 seeds were germinated for five days and then hypocotyls were exercised, cotyledons

separated and each explant was wounded near the cotyledon node. Wounded explants were co-cultivated for 5 days with

Agrobacterium tumefaciens harboring the pHL662 construct. Explants were then transferred to shoot induction (SI) medium

that contained no selection for 2 weeks and then transferred to SI medium that contained hygromycin B selection for another 2

weeks. Explants were then transferred to shoot elongation (SE) medium under hygromycin B selection for 2-8 weeks [5].

The T-DNA of this construct contains the GmSIZ1 cassette, and the hptll gene, which provides resistance to hygromycin B

antibiotic. Outside of the T-DNA border is genes that allow for selection of transgenic Agrobacterium for kanamycin and

rifampcin.

Figure 1

Sumoylation

Sumoylation is the process of post-translational modification of proteins in which small ubiquitin related modifiers (SUMOs) are altered to targeted proteins. This modification

regulates a variety of cellular processes including transcription, replication, chromosome regulation and DNA repair. SUMO protein conjugation to substrates occurs through a

cascade including E1, an activation enzyme, conjugating enzyme E2 and protein ligase E3. The modification of proteins by sumoylation can alter protein to protein

interactions, protein intracellular localization and changes to protein function [1].

Sumoylation Pathway

SUMO undergoes processing by ubiquitin-like protein specific proteases (Ulps) and sentrin-specific proteases (SENPs) before maturing. SUMO is transferred through the E1, E2 and E3 enzyme complexes and is then recycled after use for further use [1].

Figure 2

Plant Regeneration Progress

This project is still in progress. To date the transformation of explants is in the shoot elongation stage. Regenerates shoots are heathy on selection media, which is a tentative indication that

transformation and regeneration success.

Figure 3 Figure 4

Shoot Induction (SI) Shoot Elongation (SE)

OsSIZ1 Expression

Root biomass change in OsSIZ1 expression bent grass plants subjected to heat stress [3].

References [1] Gareau, J. R., & Lima, C. D. (2010). The SUMO pathway: Emerging mechanisms that shape specificity, conjugation and recognition. Nature Reviews Molecular Cell Biology Nat Rev Mol Cell Biol, 11(12), 861-871. Doi:10.1038/nrm3011[2]Hatzfeld, Yves, Christophe Reuzeau, and Valerie Frankard. “US 2012/0227133 A1 - Plants Having Enhanced Yield-Related Traits And A Method For Making The Same -  The Lens,” September 2, 2012.[3] Li, Z., Hu, Q., Zhou, M., Vandenbrink,J., Li, D.,Menchyk, N., Luo, H. (2012). Heterologous expression of OsSIZ1, a rice SUMO E3 ligase,enhances broad abiotic stress tolerance in transgenic creeping bentgrass. Plant Biotechnol J Plant Biotechnology Journal, 11(4), 432-445. doi:10.1111/pbi.12030[4]Johnson, Erica. “Protein Modification by SUMO - Annual Review of Biochemistry, 73(1):355.” Reference. Annual Reviews, March 5, 2004.[5]Zeng, P., Vadnais, D. A., Zhang, Z., & Polacco, J. C. (2004). Refined glufosinate selection in Agrobacterium -mediated transformation of soybean [Glycine max (L.) Merrill]. Plant Cell Reports, 22(7), 478-482. Doi:10.1007/s00299-003-0712-8

FundingThis project is funded by the Natural Sciences and Mathematics

Department at SUNY Cobleskill. Acknowledgements

We would like to thank Dr. Hong Luo and his lab in the Genetics and Biochemistry Department at Clemson University for

providing the pHL662 gene construct used in this experiment.

Figure 5

WT - Wild TypeTG1 - Transgenic 1TG2 – Transgenic 2

(Gareau, 2010)

(Li, 2012)