Environmental stresses of field growth allow cinnamyl alcohol dehydrogenase-deficient Nicotiana ...

68 Pages·2012·3.14 MB·English

Plant Physiology Preview. Published on June 1, 2012, as DOI:10.1104/pp.112.196717
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Environmental stresses of field growth allow cinnamyl alcohol
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dehydrogenase-deficient Nicotiana attenuata plants to compensate for their
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structural deficiencies
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Harleen Kaur,1,5 Kamel Shaker,2,6 Nicolas Heinzel,1,7 John Ralph,3 Ivan Galis1,4 and
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Ian T. Baldwin1*
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1Department of Molecular Ecology, Max-Planck Institute for Chemical Ecology, Jena
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07745, Germany; 2Department of Biosynthesis/Nuclear Magnetic Resonance, Max-
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Planck Institute for Chemical Ecology, Jena 07745, Germany; 3Department of
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Biochemistry and the DOE Great Lakes Bioenergy Research Center, University of
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Wisconsin, Madison, WI 53706, USA; 4Institute of Plant Science and Resources,
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Okayama University, Okayama 710-0046, Japan
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*Corresponding author
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Running title: Lignin deficient plants in nature
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Estimated number of printed pages: 25.8
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Footnotes:
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* Corresponding author; e-mail: [email protected]
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5,6,7 Present address: 5Boyce Thompson Institute for Plant Research, Cornell
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University, Ithaca, New York 14853, USA; 6Chemistry Department, Faculty of
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Science, King Khalid University, Abha 9004, Saudi Arabia; 7Leibniz Institute of Plant
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Genetics and Crop Plant Research (IPK), Gatersleben 06466, Germany.
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The author responsible for distribution of materials integral to the findings presented
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in this article in accordance with the policy described in the Instructions for Authors
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(www.plantcell.org) is: Ian T. Baldwin ([email protected])
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ABSTRACT
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The organized lignocellulosic assemblies of cell walls provide the structural
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integrity required for the large statures of terrestrial plants. Silencing two cinnamyl
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alcohol dehydrogenase genes (NaCADs) in Nicotiana attenuata produced plants (ir-
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CAD) with thin, red-pigmented stems, low CAD and SAD activity, low lignin contents,
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and rubbery, structurally unstable stems when grown in the glasshouse (GH).
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However, when planted into their native desert habitat, ir-CAD plants produced
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robust stems that survived wind storms as well as the wild type (WT) plants. Despite
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efficient silencing of NaCADs transcripts and enzymatic activity, field-grown ir-CAD
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plants had delayed and restricted spread of red stem pigmentation, a color change
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reflecting blocked lignification by CAD silencing, and attained WT-comparable total
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lignin contents. The rubbery GH phenotype was largely restored when field-grown ir-
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CAD plants were protected from wind, herbivore attack and UV-B exposure and
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grown in restricted rooting volumes; and conversely, lost when ir-CAD plants were
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experimentally exposed to wind, UV-B and grown in large pots in growth chambers.
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Transcript and LC-ESI-TOF analysis revealed that these environmental stresses
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enhanced the accumulation of various phenylpropanoids in stems of field-grown
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plants; GC-MS and NMR analysis revealed that the lignin of field-grown ir-CAD
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plants had GH-grown comparable levels of sinapaldehyde and syringaldehyde cross-
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linked into their lignins. Additionally, field-grown ir-CAD plants had short, thick stems
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with normal xylem element traits, which collectively enabled field-grown ir-CAD
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plants to compensate for the structural deficiencies associated with CAD silencing.
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Environmental stresses play an essential role in regulating lignin biosynthesis in
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lignin-deficient plants.
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Word count: 248
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INTRODUCTION
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Plant cell walls are chemically complex, consisting of an extra-cytosolic matrix
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composed of extensively cross-linked polysaccharides – cellulose, hemicelluloses,
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and pectin – and are impregnated with lignin and proteins (Somerville et al., 2004;
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Popper, 2008; Gille et al., 2009; Achyuthan et al., 2010; Gilbert, 2010; Linoetti et al.,
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2010; Seifert and Blaukopf, 2010). Cell wall lignification is one of the most important
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evolutionary adaptations that plants evolved to facilitate their move to terrestrial
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habitats (Weng and Chapple, 2010). In addition to contributing to structural integrity,
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mechanical support and robustness of plant stems, lignin waterproofs the cell wall
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enabling long-distance water transport and protects plants from micro-organisms,
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herbivores and oxidative stress caused by UV irradiation (Koehler and Telewski,
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2006; Robinson and Mansfield, 2009; Xu et al., 2009; Barakat et al., 2010; Kim et al.,
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2010; Tronchet et al., 2010). Lignin is chemically heterogeneous due to its
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biosynthesis by a free-radical-mediated polymerization process, which links various
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phenolic monomers with carbon-carbon as well as ether linkages, making lignin
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structurally the most recalcitrant and cohesive component of the cell wall matrix
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(Boerjan et al., 2003; Chang, 2007; Torney et al., 2007; Nadji et al., 2009; Ralph,
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2010). This cross-coupling within lignin polymers, as well as with cell wall
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polysaccharides, contributes to the structural stiffness and strength to the cell wall,
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allowing plants to attain their awesome upright statures. This tough structural
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component makes the generation of lignin-deficient and lignin-altered plants a key
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target of plant breeders and genetic engineers, as plants with altered lignin content
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and/or composition/structure, may have diminished cell wall recalcitrance, allowing
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for more cost-effective and efficient plant biomass conversion for bioethanol or other
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liquid fuels (Chen and Dixon, 2007; Himmel et al., 2007; Vanholme et al., 2010).
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The lignin composition and content can be modified in plants by silencing the
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expression of enzymes involved in the phenylpropanoid pathway. For instance,
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researchers have targeted the early up-stream enzymes such as phenylalanine
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ammonia lyase (PAL; Huang et al., 2010; Song and Wang, 2010), cinnamate-4-
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hydroxylase (C4H; Chen and Dixon, 2007), 4-coumarate-Co-A ligase (4CL; Hu et al.,
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1999; Wagner et al., 2009; Voelker et al., 2010, 2011a, 2011b), hydroxycinnamoyl-
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CoA:shikimate hydroxycinnamoyl transferase (HCT; Li et al., 2010), coumarate 3-
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hydroxylase (C3H; Ralph et al., 2006) and caffeoyl-CoA 3-O-methyltransferase
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(CCoAOMT; Day et al., 2009; Guo et al., 2001; Wagner et al., 2011) as well as the
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downstream enzymes such as cinnamoyl-CoA reductase (CCR; Ralph et al., 1998;
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Leplé et al., 2007; Derikvand et al., 2008; Wadenbäck et al., 2008; Ruel et al., 2009;
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Zhou et al., 2010) and caffeate/5-hydroxyferulate O-methyltransferase (COMT;
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Piquemal et al., 2002; Guillaumie et al., 2008; Lu et al., 2010; Tu et al., 2010) and
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cinnamyl alcohol dehydrogenase (CAD; (Halpin et al., 1994, Yahiaoui et al., 1998;
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Sibout et al., 2003, 2005). CAD is a multifunctional enzyme that regulates the last
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step of lignin biosynthesis by catalyzing the NADPH-dependent reduction of p-
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coumaraldehyde, coniferaldehyde and sinapaldehyde to their corresponding
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hydroxycinnamyl alcohol (monolignols), which are further incorporated into lignin as
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p-hydroxyphenyl (or H units), guaiacyl (or G units) and syringyl (or S units) units,
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respectively (Kim et al., 2002; Raes et al., 2003; Saballos et al., 2009).
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Upon perceiving stress, plants undergo large-scale transcriptional,
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translational and post-translational re-programming that directly influences a network
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of processes spanning the various levels of biological organization from the cellular
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to organismic levels (Hahlbrock et al., 2003; Molinier et al., 2006; Meldau et al.,
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2009; Nakashima et al., 2009; Spoel et al., 2009; Zeller et al., 2009; Walley and
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Dehesh, 2010). Lignification is also known to be stress-inducible and is influenced by
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environmental stresses such as ozone, UV-B, heavy metal exposure, drought,
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pathogen infection, nitrogen fertilization, as well as by mechanical wounding,
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herbivore attack, thigmomorphogenic responses (wind and mechanical stimulations)
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and gravity (Pomar et al., 2004; Koehler and Telewski, 2006; Saidi et al., 2009;
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Moura et al., 2010; Simmons et al., 2010; Zhao and Dixon, 2010). Field studies have
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been conducted with plants altered in their lignin contents or composition to address
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the following objectives: (i) kraft pulping and saccharification properties, (ii) the
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ecological consequences of lignin reduction for plant-herbivore and -pathogen
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interactions and decomposition, and (iii) the consequences of environmental
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stresses on morphology, physiology and wood characteristics.
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Independent glasshouse (GH) and field studies were conducted with
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antisense CAD- and COMT-silenced poplar (Populus tremula X Populus alba; Pilate
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et al., 2002; Halpin et al., 2007) and CCR- and COMT-silenced (using an RNA
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interference strategy (RNAi)) perennial ryegrass (Lolium perenne; Tu et al., 2010)
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plants. These studies revealed that growth conditions had little impact on gene
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silencing efficiency and associated enzymatic activity, lignin content and chemistry,
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plant growth, and plant-insect or plant-microbial/pathogen interactions. Poplar (~10%
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reduced Klason lignin content in one of the ir-CAD lines) and perennial ryegrass
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(~37% and ~6% reduced acetyl bromide lignin contents in CCR- and COMT-silenced
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field plants, respectively) transgenics retained their reduced lignin phenotype even
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when grown under field conditions. However, a decline in growth was noticed in
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CCR- and COMT-silenced perennial ryegrass plants, when the daily average
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temperature exceeded 40°C. Additionally, COMT-silenced perennial ryegrass plants
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experienced higher Puccinia rust infestations than did control plants in the field. In
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contrast, a decline in growth rates was observed in all CCR-silenced transgenic
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poplar lines when planted in the field (Leplé et al., 2007). The trend observed
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amongst independently CCR-silenced transgenic poplar lines found growth to be
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well correlated with the amount of lignin produced in these lines. A recent field study
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of 4CL down-regulated hybrid poplar plants found 4CL gene expression level and
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lignin chemistry to be key determinants of above-ground biomass, wood color and
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tree form (Voelker et al., 2010). Highly silenced 4CL poplar plants had reduced
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above-ground biomass, irregular cambial activity and reduced apical dominance that
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increased branching of free-standing plants in the field, and resulted in vine-like
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growth when plants were supported with stakes in the GH (Voelker, 2009). These
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studies suggest that lignin-deficient plants respond differentially to growth
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environments that could be gene-/species-specific or lignin content and chemistry
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specific responses. Little is still known about the stresses that influence lignin
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chemistry of lignin-deficient plants when grown under field conditions.
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Nicotiana attenuata is a well-studied ecological model system that has been
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used to study traits that are important for the adaptation of plants to extreme
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environmental stresses. In N. attenuata’s native habitat, the Great Basin Desert,
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Utah (USA), plants are exposed to a plethora of stresses simultaneously such as
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heavy storms, herbivore and pathogen attack, high temperature, low humidity, high
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light intensity and UV-B exposure. Here we examined the ecological dynamics of
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lignin-deprived N. attenuata plants silenced in their CAD activity in the plants’ natural
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environment. We transformed N. attenuata plants using RNAi technology with an
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inverted-repeat (ir) sequence of their endogenous CAD gene to silence two stem-
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expressed NaCADs, referred to as ir-CAD plants. The ecological importance of
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lignification in N. attenuata-Trichobaris mucorea (tobacco stem weevil) interaction in
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the field has been recently reported using these NaCAD-silenced plants (Diezel et
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al., 2011). Here, we addressed the following questions by conducting a series of
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experiments with N. attenuata ir-CAD plants grown in both the GH and the plants’
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native habitat in Utah: (i) what role does lignin play in plant development and stress
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resistance? (ii) how do the stressful conditions influence the accumulation of various
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metabolites derived from the phenylpropanoid pathway in lignin-deprived plants and
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what is their contribution to lignin composition and content? (iii) how do
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environmental stresses influence the structural strength and architecture of lignin-
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deficient plants?
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RESULTS
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Two CAD genes are silenced in ir-CAD plants
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Cinnamyl alcohol dehydrogenase1 (NaCAD1) was amongst 27 highly
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upregulated proteins that showed consistent accumulation in N. attenuata leaves
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when plants were elicited by wounding and the wounds were immediately treated
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with Manduca sexta’s oral secretion (OS) or fatty acid-amino acid conjugates (FAC,
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an elicitor present in M. sexta OS; Giri et al., 2006). This suggests that N. attenuata
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plants may enhance lignification by synthesizing more monolignols in the elicited leaf,
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perhaps to slow the entry of micro-organisms or increase leaf toughness to reduce
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further herbivory. While isolating the full-length coding sequence of NaCAD1 (a gene
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fragment isolated by Giri et al. (2006) during a proteomic analysis), PCR-based
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cloning using wounding+OS-elicited leaf cDNA as a template, resulted in the
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isolation of an additional CAD gene, which was named NaCAD. When aligned at
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nucleotide and protein levels (see Supplemental Fig. S1-S2), NaCAD and NaCAD1
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shared a very high sequence identity to each other (~85%). When a phylogenetic
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tree using all known Arabidopsis thaliana CAD genes was constructed, it revealed
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that both N. attenuata CAD genes cluster with those of A. thaliana’s bona fide CAD
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genes, namely AtCAD4 and AtCAD5, which are involved in floral stem lignification,
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suggesting that these CAD genes might also regulate lignification in N. attenuata
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(Fig. 1A; Sibout et al., 2003, 2005; Tronchet et al., 2010). Furthermore, the amino
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acid sequences of both NaCADs have well-conserved substrate-binding pockets and
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secondary structural elements (α-helices and β-strands) spanning catalytic and
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nucleotide-binding domains similar to those of AtCAD4 and AtCAD5– the
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characteristic signature of genes of the CAD family, as reported by Youn et al.
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(2006); highlighted in Supplemental Fig. S2. To assess the number of CADs in the
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N. attenuata genome, we probed a DNA blot with a fragment of NaCAD1 that had no
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restriction site for EcoRI and HindIII. After high stringency washes of the hybridized
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blots, four distinct bands were detected in the EcoRI digest and seven in the HindIII
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digest, indicating that CADs exist as a multi-gene family in N. attenuata
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(Supplemental Fig. S3A).
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We next investigated the tissue-specific transcript abundance of both CAD
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genes in wild type (WT) N. attenuata plants from multiple tissues available at the
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mature stage of plant development (Fig. 1B). Both NaCAD and NaCAD1 genes are
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strongly expressed in stems, which is consistent with their expected function in stem
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lignification (Fig. 1C, D).
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In order to functionally test the activity of both CAD proteins in vivo, we used
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an RNAi approach to silence the expression of the CAD genes in N. attenuata. The
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pRESC5CAD binary vector containing the inverted repeat (ir) fragment of the
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endogenous NaCAD1 sequence used for silencing is shown in Supplemental Fig.
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S3B. Initially twenty independently transformed T lines were screened on
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hygromycin-containing media, based on segregation analysis, 9 homozygous lines
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were selected in T generation. The number of T-DNA insertions was determined by
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Southern hybridization of genomic DNA using a PCR fragment of the hygromycin
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phosphotransferase (hptII) gene as a probe (Supplemental Fig. S3C). As expected
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from the nucleotide and protein sequence similarities between NaCAD and NaCAD1,
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NaCAD1 silencing co-silenced NaCAD in 6 of the 9 ir-CAD lines examined
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(Supplemental Fig. S3D). All six independently transformed lines that had
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significantly reduced transcript expression of both CAD genes also had weak stem
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phenotypes. For further studies two of these 6 independently transformed, diploid,
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homozygous lines harboring a single-copy of the T-DNA-insertion and strongly
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silenced in the expression of both CAD genes (both apical and basal stem: 91-93%
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silenced for NaCAD1 and 97% silenced for NaCAD in both lines) were selected for
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further experiments (ir-CAD-206 and ir-CAD-225; Inset Fig. 1A and 1B,
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Supplemental Fig. S3C; Table1).
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GH-grown ir-CAD plants develop a “rubbery” phenotype
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Lignin deposition in cell wall plays an important role in plant development,
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especially during elongation. As expected, CAD-silencing in N. attenuata produced
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plants with strongly altered stem morphologies and architectures after their
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developmental transition from rosette- to flowering-stage growth (Fig. 2A and B). In
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the initial stages of bolting (46 days after germination), the length and appearance of
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GH-grown ir-CAD and WT stems were similar (Supplemental Fig. S4A, unpaired t
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test, P = 0.087; Supplemental Fig. S4B, unpaired t test, P = 0.631).
ir-CAD-206 ir-CAD-225
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However, a delay in stem elongation was observed during the third week of bolting in
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ir-CAD plants (53 days after germination; unpaired t test, P < 0.0001; P
ir-CAD-206 ir-CAD-
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< 0.0001), which resulted in significantly shorter plants at maturity (75 days after
225
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germination; unpaired t test, P < 0.0001; P < 0.0001). A slight
ir-CAD-206 ir-CAD-225
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reduction in ir-CAD stem diameter was also observed at the base of the stem
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(Supplemental Fig. S4C; unpaired t test, P = 0.026; P = 0.025) and
ir-CAD-206 ir-CAD-225
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the upper part (40 cm from the base) of the main stem axis (unpaired t test, P
ir-CAD-206
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= 0.004; P = 0.003). Mature ir-CAD plants were bushy in appearance, having
ir-CAD-225
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more lateral branches (>15 cm in length; Fig. 2B, Supplemental Fig. S4C, D;
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unpaired t test, P = 0.008; P < 0.0001) and these branches were
ir-CAD-206 ir-CAD-225
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located closer to the base of the stems than was the case for WT plants (α;
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Supplemental Fig. S4C-E; unpaired t test, P = 0.010; P = 0.010).
ir-CAD-206 ir-CAD-225
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These lateral branches spread farther from the main stem of ir-CAD plants than from
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those of WT plants. This morphological change was quantified as the difference in
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both (a) the angle between the main stem and the lateral branches (unpaired t test,
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P < 0.0001; P < 0.0001) and (b) the distance between the main stem
ir-CAD-206 ir-CAD-225
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and a point 15 cm along the lateral branch after its attachment to the main stem (ρ;
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Supplemental Fig. S4C, E; unpaired t test, P < 0.0001; P < 0.0001).
ir-CAD-206 ir-CAD-225
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When ir-CAD plants were grown unsupported in the GH, most of the plants
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buckled under their own weight at maturity: ir-CAD stems were soft and flexible, and
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could be easily bent by hand. Because these plants were unable to grow upright and
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were structurally unstable at maturity, we refer to them as having a “rubbery”
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phenotype. We conducted one-point bending tests on basal stem segments of ir-
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CAD and WT plants to quantify their stiffness. Highly significant differences were
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observed both in the Young’s modulus and bending strength amongst ir-CAD and
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WT stems (Table1, Supplemental Fig. S5A and B), suggesting that a lack of stiffness
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of ir-CAD stems was responsible for their instability. Despite striking changes in stem
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morphologies of ir-CAD plants, no differences were observed in their fresh biomass
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(Supplemental Fig. S4C; unpaired t test, P = 0.447; P = 0.259) and
ir-CAD-206 ir-CAD-225
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seed production capacity (Supplemental Fig. S4C; unpaired t test, P = 0.583;
ir-CAD-206
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P = 0.532) when compared to the WT plants. However, ir-CAD plants had
ir-CAD-225
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less dry biomass than did WT plants (unpaired t test, P = 0.002; P <
ir-CAD-206 ir-CAD-225
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0.0001). In addition, the CAD-silenced lines tended to senesce earlier than did the
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WT plants.
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An intense reddish-brown color was first noticeable in the roots of ir-CAD
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plants and later appeared at the base of the main stems and their respective lateral
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branches; the color spread acropetally along the longitudinal axis of the stem
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replacing the normally green color of the stem as the lignification process proceeded
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(Fig. 2C and D). This red coloration was restricted to the xylem of ir-CAD stems, the
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portions that would otherwise be lignified in WT stems (Fig. 2E-G).
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To examine the degree of xylem lignification, we stained cross-sections of ir-
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CAD as well as WT stems with toluidine-blue O dye. The red xylem tissue of ir-CAD
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stems turned various shades of violet upon staining as a result of varying red
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backgrounds; however, the normally lignified tissue of WT plant developed a blue
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color (Fig. 2H-K). The red xylem of ir-CAD stems was weakly stained, poorly
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lignified, had collapsed elements (vessel and fibres) with thin, highly wavy,
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disorganized and non-coherent walls (indicated by black arrows in Fig. 2K); whereas
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the WT stems xylem had highly lignified xylem with thick-walled elements.
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Characterization of lignin and enzyme activity of GH-grown ir-CAD plants
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We examined the effect of NaCAD and NaCAD1 gene silencing on CAD/SAD
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enzymatic activity by performing in vitro assays using the corresponding alcohol
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substrates (sinapyl alcohol or coniferyl alcohol) and crude protein extracts from basal
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stem segments (0-10 cm above root/shoot transition zone) of plants at early
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elongating stages as well as from the unlignified apical part of mature stems of
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flowering-stage plants. Silencing the NaCADs yielded plants with highly reduced
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CAD (retained only ~10-15% of WT activity in both lines at both developmental
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stages) and SAD (retained only ~5-10% of WT activity) enzymatic activities (Table 1,
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Supplemental Fig. S5C and D).
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We further analyzed CAD/SAD associated changes in lignin content and
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composition of basal parts of mature WT/ir-CAD stems as WT stems are expected to
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be lignified at this stage of development and, hence, the contrasting lignin-
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associated changes of ir-CAD stems could be readily visualized. The growth
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experiments were initially conducted on two independently transformed ir-CAD lines;
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due to the similarity of the growth phenotype of both lines, the lignin analyses
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(composition and content) were performed only on the ir-CAD-206 line.
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Quantification of Klason lignin revealed that CAD-silenced N. attenuata plants had
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highly reduced (~40%) stem lignin contents relative to WT plants (unpaired t test,
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P = 0.022; Table1). N. attenuata WT plants released more thioethylated S
GH-ir-CAD
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monomeric units compared to G monomeric units from their lignin by analytical
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thioacidolysis. The amounts of released S and G monomeric units, indicative of the
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–O–4-linked units, were significantly reduced in the lignin of ir-CAD stems
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compared to that of WT stems; however, no change was observed in the release of
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H monomeric units amongst the genotypes (Table1). The reduction in S units was
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greater compared to G units in CAD-silenced plants (in ir-CAD plants relative to WT
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plants, %S units = 28%; %G units = 52%), which was also evident from the low
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thioacidolysis S:G ratio of ir-CAD plants. Apart from quantifying the most abundant
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monolignol units in the lignin polymer, the less frequent thioacidolysis-derived
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indenes from sinapaldehyde or coniferaldehyde 8–O–4-linked units, and the
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dithioketals from 4-O-end-linked syringaldehyde and vanillin units, as reported by
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Kim et al. (2002) and Lapierre et al. (2004), were recovered only from ir-CAD
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samples, highlighting the atypical incorporation of hydroxycinnamaldehydes into the
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lignin heteropolymer. It should be noted that although the hydroxycinnamaldehydes
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