Use of Cyanobacterial Proteins to Engineer New Crops

Finally, a few cyanobacterial genes involved in tolerance to environmental and/or nutritional stresses have disappeared from the plant genome during the evo-lutionary pathway from cyanob

Use of Cyanobacterial Proteins to Engineer New Crops

Matias D Zurbriggen, Néstor Carrillo, and Mohammad-Reza Hajirezaei

Abstract Cyanobacteria, the closest living relatives of the ancient endosymbiont

that gave rise to modern-day chloroplasts, offer a rich source of genes for plant genetic engineering, due to both similarities with and differences from the plant genetic systems On the one hand, cyanobacteria share many metabolic path-ways with plant cells, and especially with chloroplasts, which may be critical when the transgenic product needs to interact with endogenous systems or substrates to exert its function On the other hand, most mechanisms involved in plant regula-tion of gene expression have arisen after endosymbiosis, permitting a more raregula-tional manipulation of the introduced trait, free from host regulatory networks In addi-tion, sequence divergence between plant genes and their cyanobacterial orthologues prevents, in most cases, the unwanted consequences of gene silencing and cosup-pression Finally, a few cyanobacterial genes involved in tolerance to environmental and/or nutritional stresses have disappeared from the plant genome during the evo-lutionary pathway from cyanobacteria to vascular plants, raising the possibility of recovering these adaptive advantages by introducing those lost genes into transgenic plants In spite of their obvious potential, the use of cyanobacterial genes to engineer plants for increased productivity or stress tolerance has been relatively rare In this chapter, we review several examples in which this approach has been applied to plant genetic engineering with considerable success They include modification of central metabolic pathways to improve carbon assimilation and allocation by expressing unregulated cyanobacterial enzymes, development of chilling tolerance by increas-ing desaturation of membrane-bound fatty acids, pigment manipulation, shifts in light quality perception, production of biodegradable polymers, and synthesis of ketocarotenoids not present in crops Tolerance to adverse environments could be achieved by the introduction of cyanobacterial genes lost from the plant genome during evolution, such as flavodoxin The results obtained illustrate the power of gene and data mining in cyanobacterial genomes as a biotechnological tool for the

M.D Zurbriggen (B)

División Biología Molecular, Facultad de Ciencias Bioquímicas y Farmacéuticas,

Instituto de Biología Molecular y Celular de Rosario (IBR, UNR/CONICET),

Universidad Nacional de Rosario, S2002LRK Rosario, Argentina

e-mail: matiaszurbriggen@gmail.com

65

A Kirakosyan, P.B Kaufman, Recent Advances in Plant Biotechnology,

DOI 10.1007/978-1-4419-0194-1_4,  C Springer Science+Business Media, LLC 2009

design of transgenic plants with higher productivity, enhanced tolerance to environ-mental stress, and potential for biofarming

4.1 Introduction

Development of crops with higher productivity, nutritional value, or potential for biofarming is a major goal of plant biotechnology Direct transfer of plant genes has resulted in new varieties with improved properties However, this approach is limited by the genetic stock of extant plant species Therefore, the use of bacterial genes to engineer crop and model plants has become commonplace, with

expres-sion of the Bt toxin of Bacillus thuringiensis (milky spore bacterium) being the

most conspicuous case of worldwide application to agriculture (for a recent review, see Jube and Borthakur, 2007) There are also limitations to the use of heterolo-gous genes in transgenic plants, with important implications for the effectiveness of the desired manipulation Several factors play a role in the success of this strategy, including the expression level of the transgene in the alien environment, successful interaction with suitable endogenous partners, availability of substrate if the trans-gene product is an enzyme, compartmentalization, and codon usage In this sense, cyanobacteria offer special opportunities for crop improvement due to both impor-tant similarities with and differences from the plant genetic system With respect to the former aspect, it is worth noting that many plant metabolic, regulatory, and dissi-pative pathways, especially those concerning chloroplast physiology, were evolved from cyanobacterial ancestors “enslaved” after the successful endosymbiosis that gave origin to photosynthetic eukaryotes Many of these routes have not diverged much, thus allowing productive interactions of the transgenic products with the cor-responding endogenous systems At the same time, an unknown number of regu-latory networks that complicate handling of transgene expression are newcomers

in plant development and are not present in cyanobacteria, permitting a more cus-tomized manipulation of the engineered traits The sequence divergence between plant proteins and their cyanobacterial counterparts also prevents in most cases the undesired consequences of gene silencing and cosuppression Finally, a few genes

of cyanobacterial origin have disappeared from the plant genome or have been pro-foundly modified Their introduction into plants opens unpredictable possibilities to regain some of the adaptive advantages that allowed cyanobacteria to flourish and spread at the beginning of aerobic times on Earth

The upcoming challenge for the scientists is to use specific genes from various sources to achieve a broad tolerance of plants to rapidly changing environmental conditions In general, the final goal is to develop plants with higher yields or tol-erant to unfavorable stress situations It is also an important aim to generate plants with high-quality food properties or capable of producing renewable products (bio-farming) (Fig 4.1) Despite the many potential advantages of cyanobacterial genes, their use has still been relatively limited We review herein the various existing

Fig 4.1 Summary of current advances in plant biotechnology using cyanobacterial genes Gene

mining on cyanobacteria offers a promising opportunity as a tool for biotechnological approaches

examples, highlighting the cases in which their employment was particularly suc-cessful (Table 4.1)

4.2 Manipulation of CO2Fixation and Sugar Metabolism

Photosynthetic carbon metabolism is believed to be one major determinant for plant growth and final yield To date, huge efforts have been made to use endogenous genes in order to modify photosynthetic carbon assimilation and partitioning with the aim to improve plant productivity (Morandini and Salamini, 2003; Geigen-berger et al., 2004; Long et al., 2006) In most cases, these studies failed to provide evidence for improvement of plant biomass production, since endogenous genes

derived from higher plants are likely to be prone to fine regulation in vivo In this

connection, the main reasons could be found in the modulation of enzyme activity

by allosteric effects and covalent modification such as phosphorylation, or via inter-mediates and effectors such as glucose 6-phosphate (G6P) (Krause et al., 1998), suggesting that the use of cyanobacterial enzymes with different regulatory mech-anisms could be a promising alternative Attempts that have been made to manip-ulate photoassimilate production, sucrose metabolism, and sugar utilization will be discussed

glutamicum Synec

(aminoacid biosynthesis)

(2004) Chen

9 -desaturase

yl-lipid 9

6 -desaturase

β-Carotene k

Solanum tuber

polymers (Section

(2005) Hühns

c6

4.2.1 Carbon Assimilation

The Calvin cycle, which is the primary route for carbon assimilation in the

chloro-plasts of C3 plants (Sharkey, 1985), can be divided into three phases The first one

(RuBP) catalyzed by ribulose-1,5-bisphosphate-carboxylase/oxygenase (Rubisco),

generating 3-phosphoglycerate (3PGA) In the second phase, the reduction step, 3PGA, is converted to triose phosphate, and finally, the regeneration phase

assimi-lates formed are used either to synthesize transitory starch in the plastids or to produce sucrose in the cytosol (Quick and Neuhaus, 1997) In order to main-tain a balance between the photoassimilate export to the cytosol and the regen-eration of the acceptor molecule, RuBP, an accurate regulation of the enzymes involved in the Calvin cycle is necessary Transgenic approaches, mainly through downregulation of endogenous genes, have been performed to identify rate-limiting

fructose-1,6-bisphosphatase (FBPase), and aldolase have been reported (for details, see Frommer and Sonnewald, 1995) Based on the results obtained, the authors conclude that a successful modulation of metabolite distribution can only be achieved by using unregulated enzymes such as the plastidic aldolase Follow-ing this rationale, Miyagawa et al (2001) isolated a bifunctional unique enzyme,

fructose-1,6-/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), from Synechococ-cus PCC 7942 and demonstrated that it could hydrolyze both FBP and SBP with

almost equal specific activities The absence of homology between this enzyme and higher plants’ FBPase and/or SBPase encouraged Miyagawa and coworkers to use

it for genetic engineering Overexpression of the cyanobacterial gene in tobacco

plants (Nicotiana tabacum) under the control of the tomato rbscS promoter and

chloroplast-targeting sequence led to an increased photosynthetic capacity in source leaves, carbohydrate accumulation, and accelerated growth rate (Miyagawa et al., 2001) Recently, Tamoi et al (2006) generated transgenic tobacco plants

express-ing either Synechococcus PCC 7942 FBPase-II or Chlamydomonas SBPase in the

chloroplasts to study the individual contribution of each enzyme Interestingly, the same increase (1.6- to 1.7-fold) in the activities of either SBPase or FBPase resulted

in different outcomes While higher SBPase activity led to enhanced photosynthetic rates, FBPase overexpression failed to improve photosynthesis Using antisense RNA technology, Kossmann et al (1994) were able to show that photosynthesis and

growth rate were drastically inhibited in potato (Solanum tuberosum) plants only

when the FBPase activity was reduced below 14% of the wild-type (WT) levels

In contrast, antisense inhibition of SBPase activity strongly affected the photosyn-thetic pathway (Harrison et al., 1998, 2001) In plants with about 30% remaining SBPase activity, photosynthetic rates were diminished by 36% Collected data sug-gest that SBPase is one of the primary limiting factors for RuBP regeneration in the Calvin cycle and that an increase in its activity causes a shift toward FBPase as

a rate-limiting step of the photosynthetic carbon fixation In conclusion, the use of