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Summary
Metabolic engineering of plants allows the possibility of using crops for the synthesis of novel polymers having useful material properties. Strong and flexible protein-based polymers, which are based on the structure of silk and elastin have been synthesized in transgenic plants. A wide range of polyhydroxyalkanoates having properties ranging from stiff plastics to soft elastomers and glues have been synthesized in various compartments of plants, such as the cytoplasm, plastid and peroxisome. These plant biomaterials could replace, in part, the synthetic plastics, fibers and elastomers produced from petroleum, thus offering the advantage of renewability, sustainability and biodegradability. Key words: PHA plastic polyhydroxyalkanoate polymer silk biomaterial Abbreviations: ACP = acyl carrier protein. CaMV = cauliflower mosaic virus. CoA = coenzyme A. dwt = dry weight. HV = 3-hydroxyvalerate. MCL-PHA = medium-chain-length polyhydroxyalkanoate. PHA = polyhydroxyalkanoate. PHB = poly(3-hydroxybutyrate). P(HB-HV) = poly(3-hydroxybutyrate-co 3-hydroxyvalerate). SCL-PHA = short-chain-length polyhydroxyalkanoate. tRNA = transfer RNA
Introduction
Plants naturally synthesize a variety of polymers that have been used by humans as a useful source of biomaterials. For example, cellulose, the earth most abundant polymer and the main constituent of the plant cell wall, has been used for thousand of years as a source of fibers for various fabrics. Similarly, the latex extracted from the bark of the tree Havea brasiliensis remains a major source of rubber, despite the development of similar polymers from synthetic chemistry. In the last century, the usefulness of plant polymers as biomaterials has been expanded through their chemical modification.
* E-mail corresponding author: yves.poirier@ie-bpv.unil.ch
For example, a number of plastics have been made by substituting the hydroxyl groups present on the glucose moiety of cellulose with larger groups, such as nitrate or acetate, giving rise to materials such as cellulose acetate, a clear plastic (Nawrath et al. 1995). Similarly, starch has been used in the manufacturing of plastics by using it either in blends with synthetic polymers or as the main constituent in biodegradable plastics. Starch can be chemically modified to generate interesting polymers, such as amylose ethers, which have properties similar to polyethylene (Nawrath et al. 1995). Plant genetic engineering and the expression of foreign genes in crops has created the possibility of expanding the usefulness of natural plant biomaterials, such as through the synthesis modified starch, as well as to synthesize novel polymers in
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plants. There is thus an interest to develop transgenic plants as efficient vectors for the large scale synthesis of useful novel polymers. These plant biomaterials could replace, in part, the synthetic plastics and elastomers produced from petroleum, offering the advantage of renewability, sustainability and biodegradability. This review focuses on the use of transgenic plants for the synthesis of two novel classes of biomaterials, namely protein-based polymers made from natural or artificial genes, and polyhydroxyalkanoates, a family of polyesters having properties of biodegradable plastics and elastomers.
Protein-based biomaterials
Several naturally occurring proteins have interesting properties that function as fibers or adhesives. For example, marine mussels and some fresh water mussels, such as the zebra mussel, can produce strong protein-based adhesives that are effective in aqueous environments, allowing these organisms to stick to many surfaces and resist the action of waves, currents and tides (Yamamoto 1995). Fibrous proteins, such as elastin, collagen and silk, have remarkable material properties, including elasticity, strength, and toughness. These fibrous materials are distinct from most other proteins in that they are made of short blocks of repeated amino acids. For example, collagen is made of repeats of Gly-X-Y, where X and Y are frequently proline and hydroxyproline, while elastin is typically made of repeats of the Val-Gly-Val-Pro-Gly. Some organisms actually combine several different fibrous proteins into a single structure. For example, the marine mussel Mytilus edulis attaches to solid surfaces via a byssal thread that is stiff at one end and extensible at the other. The stiffer distal end of the thread is made of a block copolymer having domains structurally similar to silk and collagen, while the more elastic proximal end is made of a block copolymer with collagen- and elastin-like domains (Coyne et al. 1997, Qin et al. 1997). Although protein polymers lack the chemical diversity of man-made polymers, being composed of only 20 amino acids and some derivatives (such as hydroxyproline or dihydroxyphenyl--alanine), they have the advantage that their synthesis from genes allows the precise determination of both the molecular mass and the sequence of amino acids, making it possible, in principle, to achieve a degree of control over their physical properties and functionality, something which is difficult with chemical polymerisation technologies. The development and uses of protein-based biomaterials in a variety of medical or consumer products is, in many cases, limited by the difficulties in producing sufficient quantities of the material to establish structure/function relationships and design applications, as well as difficulties in their economical production for commercialisation. It is in this view that plants have been explored as a potential vector for the production of protein-based biopolymers.
Table 1. Mechanical properties of some natural and man-made biomaterialsa. Material Strength (N m 2) 4 109 1 109 4 109 1 106 1 109 Elasticity (%) 35 > 200 5 600 5
Dragline spider silk Flagelliform silk (web reinforcement) Kevlar Rubber Tendon
a
Novel biomaterials in plants Gly-X, which are separated by spacers. The amino acid composition of the spidroins is thus strongly biased toward a few amino acids, such as glycine and alanine. At the DNA level, the genes are GC-rich with blocks of repeated sequences. These features lead to a number of difficulties in the expression of these proteins in bacteria, such as Escherichia coli and Bacillus subtilis. First, the highly reiterated nature of the genes promotes recombination leading to gene deletion. Second, the high glycine and alanine content of spidroins creates problems during protein synthesis since pools of tRNA of these two amino acids are not sufficiently high to ensure adequate translation, leading to premature translation arrest and synthesis of truncated proteins. Thus, although E. coli could accumulate spidroins up to 30 % total protein from a synthetic genes which had been designed to avoid complex DNA secondary structures and have an improved codon balance, the proteins produced were highly heterogeneous in size due to gene rearrangements and premature translation arrests (Fahnestock and Irwin 1997). Surprisingly, expression of a similar gene construct in the yeast Pichia pastoris resulted in the synthesis of a full length protein up to 10 % of the total proteins, indicating that the repetitive nature of the gene construct was not necessarily an obstacle to the synthesis of these unusual proteins in eukaryotes (Fahnestock and Bedzyk 1997). Synthesis of silk proteins derived from the spiders Araneus diadematus and N. clavipes has also been achieved in mammalian cell culture (Lazaris et al. 2002). Approximalety 20 g of a 60 kD recombinant silk protein was secreted in the media per 106 cells per day, leading to the purification of 12 g of material over several months. The recombinant protein could be successfully spun into filaments showing similar toughness to dragline silk but with lower tenacity. As in E. coli, genetic instability has been observed over time and recombinant proteins larger than 60 kD were poorly expressed. Successful synthesis of high-molecular-weight spidroins has recently been demonstrated in plants (Scheller et al. 2001). Several synthetic genes containing the main repetitive peptides from N. clavipes MaSp1 protein arranged in different orders were constructed using oligonucleotides. The proteins were modified at the N- and C-terminal ends in order to direct and retain the proteins in the endoplasmic reticulum. The largest synthetic gene encoded a protein of 100 kDa. The various constructs were expressed in tobacco and potato under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The best producer plant accumulated spider silk-like proteins to greater than 2 % of the total soluble proteins. Importantly, the proteins produced were full length and no evidence of gene deletion or pre-mature translation termination was observed. The proteins could be easily purified from tobacco leaves after a few simple steps based on the heat resistance and acid solubility of the silk proteins, although production of fibers was not reported. These data showed that plants are a promising system for the large-scale production of fibrous biomaterials. Furthermore, the use of gene technology and of synthetic genes offers the opportunity to modify and tailor the
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structure of the proteins to meet specific physical properties. It is important to realize, however, that like many other fibrous proteins, the properties of silk are strongly dependant on the higher-order structure of the polymer, which itself is strongly influenced by the way the material is spun (Vollrath and Knight 2001). Thus, development and application of novel silk-like fibers produced in plants will also depend on advances in artificial spinning technologies.
Polyhydroxyalkanoates
Polyhydroxyalkanotes (PHAs) are polyesters of hydroxyacids that are synthesized in over 100 different genera of bacteria,
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Laurence Moire, Enea Rezzonico, Yves Poirier PHAs are biodegradable plastics, with several bacteria and some fungi capable of metabolising extracellular PHA to carbon dioxide and water (Jendrossek 2001). In addition of their degradability, many PHAs are also biocompatible, since the breakdown products are 3-hydroxyacids, which are naturally found in animals. This makes PHA useful in some medical applications, such as implants, gazes and suture filaments. Synthesis of PHAs in native or recombinant bacteria through fermentation is a rather expensive process, costing approximately 2 4 $/kg, making bacterial PHA 510 times more expensive than petroleum-based polymers, such as polypropylene. This limits the use of bacterial PHAs to highvalue products, such as medical applications, and excludes them as substitutes for plastics in low-value consumer products, such as packaging material and disposable items. It is in this context that agriculture has been regarded as a promising alternative for the production of PHAs on a large scale and at low cost (Poirier et al. 1995, Poirier 1999). Synthesis of PHA in crops fits into a larger concept of using plants as vectors for the renewable and sustainable synthesis of carbon building blocks that are presently largely provided by the petrochemical industry.
encompassing Gram-positive and Gram-negative species, phototrophic bacteria and cyanobacteria (Kim and Lenz 2001, Steinbchel 1991). PHAs are synthesized in bacteria as a carbon and energy reserve. They typically accumulate when bacteria are grown in media that are limited in a nutrient essential for growth (typically nitrogen or phosphorus) but with an abundant supply of carbon (e.g. glucose) (Steinbchel and Hein 2001). Under these conditions, bacteria convert the extracellular carbon into PHA, which accumulates as intracellular inclusions of 0.2 0.5 m in diameter. When the limiting nutrient is re-supplied, intracellular PHA is degraded and the resulting carbon is used for growth. PHAs represent a large group of polyesters that includes over 150 different hydroxyacids (Steinbchel and Valentin 1995). Although the majority of PHAs are composed of R-3hydroxyacids ranging from 3 to 16 carbons in length (Fig. 1), some polymers also contain monomers of 4-hydroxy and 5-hydroxyacids. Furthermore, a diversity of functional groups have been found on the side chain of the monomers forming PHAs, including unsaturated groups, as well as halogenated, cyano, and phenoxy groups, just to name a few. The majority of bacteria synthesizing PHAs can be broadly subdivided into two groups. One group produces short-chain-length PHAs (SCL-PHAs) with monomers ranging from 3 to 5 carbons in length, while a distinct group synthesizes medium-chainlength PHAs (MCL-PHAs) with monomers from 6 to 16 carbons. This classification is, however, not strict as a few bacteria have been shown to produce hybrid PHAs with monomers ranging from 4 to 10 carbons or higher (Fukui et al. 1998). The large diversity of monomers found in PHAs translates into a wide spectrum of physical properties, making them interesting biomaterials (de Koning 1995). The homopolymer poly(3-hydroxybutyrate) (PHB), the most common and well studied PHA, is a relatively stiff and brittle plastic with limited commercial applications. In contrast, PHA co-polymers composed primarily of 3-hydroxybutyrate with a fraction of longer chain monomers, such as 3-hydroxyvalerate, 3-hydroxyhexanoate or 3-hydroxyoctanoate, are more flexible and tougher plastics. These PHAs can be used in a wide variety of consumer products, such as containers and bottles as well as water-resistant films. PHAs made of longer monomers, such as MCL-PHAs, are typically elastomers and sticky materials which can also be modified to make rubbers.
Figure 1. Chemical structure of some of the 3-hydroxyacids that can be included into PHA. The monomers can range from 3 to 16 carbons in length, depending on the size of the pendant R group.
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Figure 2. Synthesis of PHA in the cytoplasm, plastid or peroxisome of plant cells. The bacterial enzymes expressed in plants cells are indicated in italic. PDC refers to the endogenous plant pyruvate dehydrogenase complex.
Figure 3. Accumulation of PHB in the chloroplast of a transgenic A. thaliana cell. PHB is seen as agglomerations of electron-lucent granules in the stroma of the chloroplast.
14 % of the leaf dry weight. Transmission electron micrograph revealed that the plastids contained numerous inclusions typical of PHB granules (Fig. 3). The accumulation of PHB in
leaves was found to increase over the life span of the plant, with fully expanded pre-senescing leaves typically showing ten times more PHB than younger expanding leaves of the same plant. Although these transgenic plants showed little signs of reduced growth, chlorosis of leaves was observed, indicating that some aspect of chloroplast function was affected. A further increase in the level of PHB accumulation in the plastid has recently been demonstrated by Bohmert et al. (2000) by combining the same three bacterial genes involved in PHB synthesis on a single binary vector. A. thaliana plants accumulating PHB as high as 40 % of the shoot dwt were detected. In these plants, progressively stronger negative effects on growth were observed for plants accumulating more than 3 % PHB dwt. Analysis of over 60 metabolites in these plants revealed a large decrease in isocitrate and fumarate. This may indicate a reduction in tricarboxylic acid cycle activity, leading perhaps to a reduction in pools of acetylCoA that may result in growth retardation. There was also a positive correlation between PHB accumulation and levels of
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Laurence Moire, Enea Rezzonico, Yves Poirier branched amino acid biosynthetic pathway (Fig. 2) (Slater et al. 1999). An E. coli threonine deaminase modified for plastid targeting was over-expressed in plants, leading to the accumulation of 2-ketobutyrate, which can be converted, albeit at low efficiency, to propionyl-CoA by the plant pyruvate dehydrogenase complex. A novel 3-ketothiolase from R. eutropha showing high affinity for both propionyl-CoA and acetyl-CoA was also targeted to the plastids along with the acetoacetyl-CoA reductase and the PHA synthase (Slater et al. 1998). Expression of all four genes in A. thaliana under the control of the constitutive CaMV35S promoter resulted in the accumulation of P(HB-HV) between 0.2 0.8 % dwt in shoots, with a 417 mol % HV content in the polymer. Seed-specific expression of the same pathway in leukoplast of rape embryos resulted in PHA accumulation up to 2.3 % dwt in seeds, with up to 6.5 mol % HV (Slater et al. 1999). Levels of P(HBHV) copolymer production are substantially lower than for PHB, indicating a potential metabolic penalty of propionylCoA formation and/or threonine deaminase expression in the plastid. Nevertheless, synthesis of BiopolTM in plants is an important milestone on the route to commercialisation of PHAproducing crops.
several sugars such as mannitol, glucose, fructose and sucrose. Together, these data indicate that high amounts of PHB accumulation in chloroplasts, has a negative effect on plant metabolism and that although the plastid was a much better site for the synthesis of PHB than the cytoplasm, accumulation of high amounts of PHB in such a vital organelle as the chloroplast has its limits. Efforts to bring the synthesis of PHB in plants to the field have been mainly pursued by scientists at Monsanto. Transformation of corn with gene constructs aimed at the synthesis of PHB in the chloroplasts of leaves and stalks resulted in accumulation of PHB up to 5.7% dwt (Mitsky et al. 2000, Poirier and Gruys 2001). Similar to the results obtained with A. thaliana, there was a correlation between leaf chlorosis and higher amount of PHB. The same group have also targeted the synthesis of PHB to the seed leukoplast of rape, resulting in accumulation of PHB up to 8 % of the mature seed dwt (Houmiel et al. 1999). These transgenic seeds had a normal size, appearance and germination frequency, indicating that the seed leucoplast may be metabolically more tolerant to the diversion of acetyl-CoA and PHA accumulation compared to the chloroplasts of green tissues. Acetyl-CoA, the central building block of PHB, is found not only in the cytoplasm and plastid, but also in the mitochondria and peroxisomes, being implicated in these organelles in the tricarboxylic acid and -oxidation cycles, respectively. Although no demonstration of PHB synthesis in plant mitochondria has been reported, synthesis of up 2 % dwt PHB was shown in transgenic Black Mexican Sweet corn suspension cell cultures expressing the PHB biosynthetic pathway in the peroxisomes (Fig. 2) (Hahn et al. 1999). No analysis of the effects of peroxisomal PHB synthesis on metabolism of this cell culture has been reported. Further experiments are needed to explore the potential advantages and disadvantages of synthesizing PHB in the peroxisomes of plants growing in the field.
Novel biomaterials in plants plant -oxidation pathway was capable of generating a large spectrum of monomers from fatty acids that can be included in MCL-PHAs. In a further extension of the study of MCL-PHA synthesis in plant peroxisome, it has been shown that an endogenous control over the flux of substrates towards -oxidation and PHA synthesis could be achieved through the combined expression of a peroxisomal PHA synthase with an acyl-ACP thioesterase (Mittendorf et al. 1999). Expression of a plastidial caproyl-ACP thioesterase from Cuphea lanceolata in A. thaliana was used to channel decanoic acids towards peroxisomal -oxidation, resulting in an 8-fold increase in synthesis of MCL-PHA in mature leaves, with the polymer being composed of approximately 40 mol % 3-hydroxydecanoic acid, 32 mol % hydroxyoctanoic acid and 4 mol % hydroxyhexanoic acid. This strategy initially applied to the synthesis of MCLPHAs in vegetative green tissues was further extended to the synthesis of PHA in developing seeds of A. thaliana (Poirier et al. 1999). In dry seeds, a maximal amount of 0.1 % dwt PHA was detected. From these studies, a working model was established whereby enzymes and genes involved in the synthesis of unusual fatty acids in plants can be used to modulate the quantity and quality of substrates channeled towards MCL-PHAs. The absolute amount of MCL-PHA accumulating in mature shoots or seeds of transgenic plants expressing both the thioesterase and the PHA synthase remains relatively low ( 0.6 % dwt) compared to PHB synthesis in plastids. The reasons for this difference could be multiple, including differential activity of bacterial enzymes in various plant subcellular compartments and the relative ability of the created and endogenous metabolic pathways to compete for the same substrates (acetyl-CoA for PHB and 3-hydroxyacylCoAs for MCL-PHAs). It is thus clear that further metabolic engineering will be necessary in order to increase the level of MCL-PHA synthesized in tissues via the peroxisome to levels 15 % dwt. The recent demonstration of the synthesis of low amount of MCL-PHA in peroxisomes of Saccharomyces cerevisiae and P. pastoris will enable the use of the powerful tools of yeast genetics and genomics to achieve this goal (Poirier et al. 2001, 2002).
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to significantly decrease the rate of heat uptake and cooling of the fiber, resulting in a higher heat capacity and improving the fiber insulating properties. Interestingly, in contrast to the large increase in accumulation of PHB in leaves of A. thaliana expressing the pathway in the chloroplast, expression of the PHB pathway in the plastid of the cotton fiber resulted in a lower amount of polymer, indicating a difference either in the number or metabolic activity of the plastids in fiber cells compared to leaves (John 1997).
Conclusions
A spectrum of PHAs ranging from the stiff PHB to the more flexible P(HB-HV) plastic and MCL-PHA elastomers have now been successfully synthesized in plants using various metabolic pathways. These experiments have shown that in some cases very high polymer yields can be obtained (Bohmert et al. 2000). There can be, however, a considerable metabolic cost to such synthesis. The challenge for the future is to succeed in the accumulation of adequate amounts of PHA ( 15 % dwt) without lowering the overall yield of other plant products, such as oils, proteins or starch. This is important since in contrast to the production of PHA by bacterial fermentation, where the system is designed to produce mainly PHA with little residual waste, a large-scale agricultural production of PHA is likely to be only viable through the recovery of not only PHA, but also of all other valuable components of the crop (Poirier 2001). For example, in the case of an oil crop such as B. napus, one must be able to recover PHA and the oil, and still be able to use the de-lipidised protein-rich meal for animal feed. In the case of a carbohydrate-producing crop such as sugar beet or sugarcane, both sucrose and PHA would have to be recovered. We know thus far that PHB can be produced in the seed of rape to 8 % dwt without obvious deleterious effects on plant growth and germination (Houmiel et al. 1999). Thus, the goal of producing adequate levels of PHA in crops without yield penalty appears realistic. Initial experiments with expression of protein-based polymers in plants indicate that, like PHAs, plants can be valuable vectors for the synthesis of these biomaterial. Further metabolic engineering of various metabolic pathways, such as amino acid biosynthesis and the manipulation of tRNA pools, is likely to contribute to further improvement in protein-polymer synthesis. The success of using transgenic plants as a source of novel material will not only depend on the production levels achieved, but also on whether the polymers can be extracted efficiently, economically and ecologically from crops. Although a number of strategies have been described in the literature for the extraction of both PHA and protein-based polymers, further work is required to validate these extraction processes in the context of large scale production in plants (Poirier 2001).
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