Research Items (20)
- Feb 2019
The Mediator complex functions as a hub for transcriptional regulation. MED5, an Arabidopsis Mediator tail subunit, is required for maintaining phenylpropanoid homeostasis. A semi‐dominant mutation (ref4‐3) that causes a single amino acid substitution in MED5b functions as a strong suppressor of the pathway, leading to decreased soluble phenylpropanoid accumulation, reduced lignin content and dwarfism. By contrast, loss of MED5 results in increased levels of phenylpropanoids. We used a reverse genetic approach to identify suppressors of ref4‐3 and found that ref4‐3 requires CDK8, a kinase module subunit of Mediator, to repress plant growth. The genetic interaction between MED5 and CDK8 was further characterized using mRNA‐sequencing (RNA‐seq) and metabolite analysis. Growth inhibition and suppression of phenylpropanoid metabolism can be genetically separated in ref4‐3 by elimination of CDK8 kinase activity; however, the stunted growth of ref4‐3 is not dependent on the phosphorylation event introduced by the G383S mutation. In addition, rather than perturbation of lignin biosynthesis, mis‐regulation of DJC66, a gene encoding a DNAJ protein, is involved in the dwarfism of the med5 mutants. Together, our study reveals genetic interactions between Mediator tail and kinase module subunits and enhances our understanding of dwarfing in phenylpropanoid pathway mutants. This article is protected by copyright. All rights reserved.
Background Monolignol-like molecules can be integrated into lignin along with conventional monolignol units, and it has been shown that the incorporation of non-canonical subunits can be used to generate hydrolysable lignin by introduction of ester linkages into the polymer and that this type of lignin is more easily removable. Disinapoyl esters (DSEs), which to some degree resemble the monolignol sinapyl alcohol, may be promising lignin modifying units for this purpose. As a first step toward determining whether this goal is achievable, we manipulated metabolic flux in Arabidopsis to increase the amounts of DSEs by overexpressing sinapoylglucose:sinapoylglucose sinapoyltransferase (SST) which produces two main DSEs, 1,2-disinapoylglucose, and another compound we identify in this report as 3,4-disinapoyl-fructopyranose. ResultsWe succeeded in overproducing DSEs by introducing an SST-overexpression construct into the sinapoylglucose accumulator1 (sng1-6) mutant (SST-OE sng1-6) which lacks several of the enzymes that would otherwise compete for the SST substrate, sinapoyglucose. Introduction of cinnamyl alcohol dehydrogenase-c (cad-c) and cad-d mutations into the SST-OE sng1-6 line further increased DSEs. Surprisingly, a reduced epidermal fluorescence (ref) phenotype was observed when SST-OE sng1-6 plants were evaluated under UV light, which appears to have been induced by the sequestration of DSEs into subvacuolar compartments. Although we successfully upregulated the accumulation of the target DSEs, we did not find any evidence showing the integration of DSEs into the cell wall. Conclusions Our results suggest that although phenylpropanoid metabolic engineering is possible, a deeper understanding of sequestration and transport mechanisms will be necessary for successful lignin engineering through this route.
Drought stress is a condition that in specific climate contexts, results in insufficient water availability and often limits plant productivity through perturbing development and reducing plant growth and survival. Plants use senescence of old leaves and dormancy of buds and seeds to survive extreme environmental conditions. The plant hormone abscisic acid (ABA) accumulates after drought stress, and increases plant survival by inducing quick responses such as stomatal closure, and long-term responses such as extended growth inhibition, osmotic regulation, accumulation of cuticular wax, senescence, abscission, and dormancy. Here we focus on how the long-term ABA responses contribute to plant survival during severe drought stress. Leaf senescence and abscission of older leaves reduce total plant transpirational water loss and increase the transfer of nutrients to meristems and to some storage tissues. Osmotic regulation favors water consumption in sink tissues, and accumulation of cuticular wax helps to seal the plant surface and limits non-stomatal water loss.
Background: Coordination of synthesis and assembly of the polymeric components of cell walls is essential for plant growth and development. Given the degree of co-mingling and cross-linking among cell wall components, cellulose organization must be dependent on the organization of other polymers such as lignin. Here we seek to identify aspects of that codependency by studying the structural organization of cellulose fibrils in stems from Arabidopsis plants harboring mutations in genes encoding enzymes involved in lignin biosynthesis. Plants containing high levels of G-lignin, S-lignin, H-lignin, aldehyde-rich lignin, and ferulic acid-containing lignin, along with plants with very low lignin content were grown and harvested and longitudinal sections of stem were prepared and dried. Scanning X-ray microdiffraction was carried out using a 5-micron beam that moved across the sections in 5-micron steps and complete diffraction patterns were collected at each raster point. Approximately, 16,000 diffraction patterns were analyzed to determine cellulose fibril orientation and order within the tissues making up the stems. Results: Several mutations-most notably those exhibiting (1) down-regulation of cinnamoyl CoA reductase which leads to cell walls deficient in lignin and (2) defect of cinnamic acid 4-hydroxylase which greatly reduces lignin content-exhibited significant decrease in the proportion of oriented cellulose fibrils in the cell wall. Distinctions between tissues were maintained in all variants and even in plants exhibiting dramatic changes in cellulosic order the trends between tissues (where apparent) were generally maintained. The resilience of cellulose to degradative processes was investigated by carrying out the same analysis on samples stored in water for 30 days prior to data collection. This treatment led to significant loss of cellulosic order in plants rich in aldehyde or H-lignin, less change in wild type, and essentially no change in samples with high levels of G- or S-lignin. Conclusions: These studies demonstrate that changes in lignin biosynthesis lead to significant disruption in the orientation and order of cellulose fibrils in all tissues of the stem. These dramatic phenotypic changes, in mutants with lignin rich in aldehyde or H-units, correlate with the impact the mutations have on the enzymatic degradation of the plant cell wall.
- Jun 2016
Lignin plays important biological functions in plant cell walls, but also contributes to the recalcitrance of the walls to deconstruction. In recent years, genetic modification of lignin biosynthesis pathways has become one of the primary targets of plant cell wall engineering. In this study, we used a combination of approaches to characterize the structural and compositional features of wild-type Arabidopsis and mutants with distinct lignin monomer compositions: fah1-2 (Guaiacyl, G-lignin dominant), C4H-F5H (Syringyl, S-lignin dominant), COMT1 (G/5-hydroxy G-lignin dominant), and a newly developed med5a med5b ref8 (p-hydroxyphenyl, H-lignin dominant) mutant. In order to understand how lignin modification affects biomass recalcitrance, substrate reactivity and lignin fractionation, we correlated these properties with saccharification efficiency after ionic liquid (IL) pretreatment. Results showed that the cleavage of ?-O-4 linkages in the H- or S-lignin mutants was greater than that in G-lignin mutants. Furthermore, density functional theory (DFT) based calculations indicate higher chemical reactivity of the linkages between H- and S-lignin monomers, a possible cause of the reduced recalcitrance of H- or S-lignin mutants. Glycome profiling was conducted to study the impact of lignin modification on overall composition, extractability, integrity and lignin-associated features of most major non-cellulosic cell wall glycans in these mutants. This study provides insights into the role of lignin monomer composition on the enzymatic digestibility of biomass and the effect of lignin modification on overall wall structure and biomass pretreatment performance.
The biosynthesis of lignin, flavonoids and hydroxycinnamoyl esters share the first three enzymatic steps of the phenylpropanoid pathway. The last shared step is catalyzed by 4-coumarate: CoA ligase (4CL), which generates p-coumaroyl CoA and caffeoyl CoA from their respective acids. Four isoforms of 4CL have been identified in Arabidopsis thaliana. Phylogenetic analysis reveals that 4CL1, 4CL2 and 4CL4 are more closely related to each other than to 4CL3, suggesting that the two groups may serve different biological functions. Promoter-GUS analysis shows that 4CL1 and 4CL2 are expressed in lignifying cells. In contrast, 4CL3 is expressed in a broad range of cell types and 4CL3 has acquired a distinct role in flavonoid metabolism. Sinapoylmalate, the major hydroxycinnamoyl ester found in Arabidopsis, is greatly reduced in a 4cl1 4cl3 mutant, showing that 4CL1 and 4CL3 function redundantly in its biosynthesis. 4CL1 accounts for the majority of the total 4CL activity and loss of 4CL1 leads to reduction in lignin content but no growth defect. The 4cl1 4cl2 and the 4cl1 4cl2 4cl3 mutants are both dwarf but do not have further reduced lignin than the 4cl1 mutant, indicating that one of 4CL1 or 4CL2 is required for normal plant growth. Although 4CL4 has a limited expression profile, it does make a modest contribution to lignin biosynthesis. Together, these data show that the four isoforms of 4CL in Arabidopsis have overlapping yet distinct roles in phenylpropanoid metabolism.
YUCCA (YUC) proteins constitute a family of flavin monooxygenases (FMOs), with an important role in auxin (IAA) biosynthesis. Here we report that Arabidopsis plants overexpressing YUC6 display enhanced IAA-related phenotypes and exhibit improved drought stress tolerance, low rate of water loss and controlled ROS accumulation under drought and oxidative stresses. Co-overexpression of an IAA-conjugating enzyme reduces IAA levels but drought stress tolerance is unaffected, indicating that the stress-related phenotype is not based on IAA overproduction. YUC6 contains a previously unrecognized FAD- and NADPH-dependent thiol-reductase activity (TR) that overlaps with the FMO domain involved in IAA biosynthesis. Mutation of a conserved cysteine residue (Cys-85) preserves FMO but suppresses TR activity and stress tolerance, whereas mutating the FAD- and NADPH-binding sites, that are common to TR and FMO domains, abolishes all outputs. We provide a paradigm for a single protein playing a dual role, regulating plant development and conveying stress defence responses.
- Aug 2015
Highly lignified vascular plant cell walls represent the majority of cellulosic biomass. Complete release of the biomass to deliver renewable energy by physical, chemical and biological pretreatments is challenging due to the "protection" provided by polymerized lignin, and as such, additional tools to monitor lignin deposition and removal during plant growth and biomass deconstruction would be of great value. We developed a hyperspectral stimulated Raman scattering microscope with 9 cm(-1) spectral resolution and submicron spatial resolution. Using this platform, we mapped the aromatic ring of lignin, aldehyde, and alcohol groups in lignified plant cell walls. By multivariate curve resolution of the hyperspectral images, we uncovered a spatially distinct distribution of aldehyde and alcohol groups in the thickened sec-ondary cell wall. These results collectively contribute to a deeper understanding of lignin chemical composition in plant cell wall.
- May 2015
Plants produce an array of metabolites (including lignin monomers and soluble UV-protective metabolites) from phenylalanine through the phenylpropanoid biosynthetic pathway. A subset of plants, including many related to Arabidopsis thaliana, synthesizes glucosinolates, nitrogen- and sulfur-containing secondary metabolites that serve as components of a plant defense system that deters herbivores and pathogens. Here, we report that the Arabidopsis thaliana reduced epidermal fluorescence5 (ref5-1) mutant, identified in a screen for plants with defects in soluble phenylpropanoid accumulation, has a missense mutation in CYP83B1 and displays defects in glucosinolate biosynthesis and in phenylpropanoid accumulation. CYP79B2 and CYP79B3 are responsible for the production of the CYP83B1 substrate indole-3-acetaldoxime (IAOx), and we found that the phenylpropanoid content of cyp79b2 cyp79b3 and ref5-1 cyp79b2 cyp79b3 plants is increased compared with the wild type. These data suggest that levels of IAOx or a subsequent metabolite negatively influence phenylpropanoid accumulation in ref5 and more importantly that this crosstalk is relevant in the wild type. Additional biochemical and genetic evidence indicates that this inhibition impacts the early steps of the phenylpropanoid biosynthetic pathway and restoration of phenylpropanoid accumulation in a ref5-1 med5a/b triple mutant suggests that the function of the Mediator complex is required for the crosstalk. © 2015 American Society of Plant Biologists. All rights reserved.
Current biomass utilization processes do not make use of lignin beyond its heat value. Here we report on a bimetallic Zn/Pd/C catalyst that converts lignin in intact lignocellulosic biomass directly into two methoxyphenol products, leaving behind the carbohydrates as a solid residue. Genetically modified poplar enhanced in syringyl (S) monomer content yields only a single product, dihydroeugenol. Lignin-derived methoxyphenols can be deoxygenated further to propylcyclohexane. The leftover carbohydrate residue is hydrolyzed by cellulases to give glucose in 95% yield, which is comparable to lignin-free cellulose (solka floc). New conversion pathways to useful fuels and chemicals are proposed based on the efficient conversion of lignin into intact hydrocarbons.
Lignin is a phenylpropanoid-derived heteropolymer important for the strength and rigidity of the plant secondary cell wall. Genetic disruption of lignin biosynthesis has been proposed as a means to improve forage and bioenergy crops, but frequently results in stunted growth and developmental abnormalities, the mechanisms of which are poorly understood. Here we show that the phenotype of a lignin-deficient Arabidopsis mutant is dependent on the transcriptional co-regulatory complex, Mediator. Disruption of the Mediator complex subunits MED5a (also known as REF4) and MED5b (also known as RFR1) rescues the stunted growth, lignin deficiency and widespread changes in gene expression seen in the phenylpropanoid pathway mutant ref8, without restoring the synthesis of guaiacyl and syringyl lignin subunits. Cell walls of rescued med5a/5b ref8 plants instead contain a novel lignin consisting almost exclusively of p-hydroxyphenyl lignin subunits, and moreover exhibit substantially facilitated polysaccharide saccharification. These results demonstrate that guaiacyl and syringyl lignin subunits are largely dispensable for normal growth and development, implicate Mediator in an active transcriptional process responsible for dwarfing and inhibition of lignin biosynthesis, and suggest that the transcription machinery and signalling pathways responding to cell wall defects may be important targets to include in efforts to reduce biomass recalcitrance.
The phenylpropanoid pathway is responsible for the biosynthesis of diverse and important secondary metabolites including lignin and flavonoids. The ref8 mutant of Arabidopsis thaliana, which is defective in a lignin biosynthetic enzyme p-coumaroyl shikimate 3'-hydroxylase (C3'H), exhibits severe dwarfism and sterility. To better understand the impact of perturbation of phenylpropanoid metabolism on plant growth, we generated a chemically-inducible C3'H expression construct and transformed it into the ref8 mutant. Application of dexamethasone to these plants greatly alleviates the dwarfism and sterility and substantially reverses the biochemical phenotypes of ref8 plants including the reduction of lignin content and hyper-accumulation of flavonoids and p-coumarate esters. Induction of C3'H expression at different developmental stages has distinct impacts on plant growth. Although early induction effectively restored the elongation of primary inflorescence stem, application to 7-week-old plants enabled them to produce new rosette inflorescence stems. Examination of hypocotyls of these plants revealed normal vasculature in the newly formed secondary xylem, presumably restoring water transport in the mutant. The ref8 mutant accumulates higher levels of salicylic acid (SA) than wild type, but depletion of this compound in ref8 did not relieve the mutant's growth defects, suggesting that the hyper-accumulation of SA is unlikely to be responsible for dwarfism in this mutant.
- Sep 2013
The Arabidopsis stem is composed of five tissues - the pith, xylem , phloem, cortex and epidermis - each of which fulfills specific roles in support of the growth and survival of the organism. The lignocellulosic scaffolding of cell walls is specialized to provide optimal support for the diverse functional roles of these layers, but little is known about this specialization. X-ray scattering can be used to study this tissue-specific diversity because the cellulosic components of the cell walls give rise to recognizable scattering features interpretable in terms of the underlying molecular architecture and distinct from the largely unoriented scatter from other constituents. Here we use scanning x-ray microdiffraction from thin sections to characterize the diversity of molecular architecture in the Arabidopsis stem and correlate that diversity to the functional roles the distinct tissues of the stem play in the growth and survival of the organism.
Figure S1. Transverse sections (60 μm) of a Pinus radiata stem stained with tetramethylbenzidine (TMB), exhibiting the sites where phenol oxidase activities were detected before (left) and after (right) heat treatment (85°C for 1 h). Figure S2. A close comparison of labeled and unlabeled sections of Pinus radiata stem at identical exposure showing the contribution of autofluorescence. Figure S3. Arabidopsis thaliana protoplast cells treated with (left) and without (right) NBD-tagged monolignol (3G, 50 μm for 4 h). Data S1. NMR spectra of synthetic compounds. Appendix S1. Synthetic procedures.
Lignin is an abundant phenylpropanoid polymer produced by the oxidative polymerization of p-hydroxycinnamyl alcohols (monolignols). Lignification, i.e., deposition of lignin, is a defining feature of secondary cell wall formation in higher plants, and provides an important mechanism for their disease resistance; however, many aspects of the cell wall lignification process remain unclear partly because of a lack of suitable imaging methods to monitor the process in vivo. In this study, a set of monolignol analogs γ-linked to fluorogenic aminocoumarin and nitrobenzofuran dyes were synthesized and tested as imaging probes to visualize the cell wall lignification process in Arabidopsis thaliana and Pinus radiata under various feeding regimens. In particular, we demonstrate that the fluorescence-tagged monolignol analogs can penetrate into live plant tissues and cells, and appear to be metabolically incorporated into lignifying cell walls in a highly specific manner. The localization of the fluorogenic lignins synthesized during the feeding period can be readily visualized by fluorescence microscopy and is distinguishable from the other wall components such as polysaccharides as well as the pre-existing lignin that was deposited earlier in development. This article is protected by copyright. All rights reserved.
Indole-3-acetic acid (IAA), a major plant auxin, is produced in both tryptophan-dependent and tryptophan-independent pathways. A major pathway in Arabidopsis thaliana generates IAA in two reactions from tryptophan. Step one converts tryptophan to indole-3-pyruvic acid (IPA) by tryptophan aminotransferases followed by a rate-limiting step converting IPA to IAA catalyzed by YUCCA proteins. We identified eight putative StYUC (Solanum tuberosum YUCCA) genes whose deduced amino acid sequences share 50 % to 70 % identity with those of Arabidopsis YUCCA proteins. All include canonical, conserved YUCCA sequences: FATGY motif, FMO signature sequence, and FAD-binding and NADP-binding sequences. In addition, five genes were found with ~50% amino acid sequence identity to Arabidopsis tryptophan aminotransferases. Transgenic potato (Solanum tuberosum cv Jowon) constitutively overexpressing Arabidopsis AtYUC6 displayed high auxin phenotypes such as narrow downward curled leaves, increased height, erect stature and longevity. Transgenic potato plants overexpressing AtYUC6 showed enhanced drought tolerance based on reduced water loss. The phenotype was correlated with reduced levels of reactive oxygen species in leaves. The results suggest a functional YUCCA pathway of auxin biosynthesis in potato that may be exploited to alter plant responses to the environment.
The Arabidopsis thaliana YUCCA family of flavin monooxygenase proteins catalyses a rate-limiting step in de novo auxin biosynthesis. A YUCCA6 activation mutant, yuc6-1D, has been shown to contain an elevated free IAA level and to display typical high-auxin phenotypes. It is reported here that Arabidopsis plants over-expressing YUCCA6, such as the yuc6-1D activation mutant and 35S:YUC6 transgenic plants, displayed dramatic longevity. In addition, plants over-expressing YUCCA6 exhibited classical, delayed dark-induced and hormone-induced senescence in assays using detached rosette leaves. However, plants over-expressing an allele of YUCCA6, that carries mutations in the NADPH cofactor binding site, exhibited neither delayed leaf senescence phenotypes nor phenotypes typical of auxin overproduction. When the level of free IAA was reduced in yuc6-1D by conjugation to lysine, yuc6-1D leaves senesced at a rate similar to the wild-type leaves. Dark-induced senescence in detached leaves was accompanied by a decrease in their free IAA content, by the reduced expression of auxin biosynthesis enzymes such as YUCCA1 and YUCCA6 that increase cellular free IAA levels, and by the increased expression of auxin-conjugating enzymes encoded by the GH3 genes that reduce the cellular free auxin levels. Reduced transcript abundances of SAG12, NAC1, and NAC6 during senescence in yuc6-1D compared with the wild type suggested that auxin delays senescence by directly or indirectly regulating the expression of senescence-associated genes.
Auxin plays critical roles in many aspects of plant growth and development. Although a number of auxin biosynthetic pathways have been identified, their overlapping nature has prevented a clear elucidation of auxin biosynthesis. Recently, Arabidopsis (Arabidopsis thaliana) mutants with supernormal auxin phenotypes have been reported. These mutants exhibit hyperactivation of genes belonging to the YUCCA family, encoding putative flavin monooxygenase enzymes that result in increased endogenous auxin levels. Here, we report the discovery of fertile dominant Arabidopsis hypertall1-1D and hypertall1-2D (yucca6-1D, -2D) mutants that exhibit typical auxin overproduction phenotypic alterations, such as epinastic cotyledons, increased apical dominance, and curled leaves. However, unlike other auxin overproduction mutants, yucca6 plants do not display short or hairy root phenotypes and lack morphological changes under dark conditions. In addition, yucca6-1D and yucca6-2D have extremely tall (>1 m) inflorescences with extreme apical dominance and twisted cauline leaves. Microarray analyses revealed that expression of several indole-3-acetic acid-inducible genes, including Aux/IAA, SMALL AUXIN-UP RNA, and GH3, is severalfold higher in yucca6 mutants than in the wild type. Tryptophan (Trp) analog feeding experiments and catalytic activity assays with recombinant YUCCA6 indicate that YUCCA6 is involved in a Trp-dependent auxin biosynthesis pathway. YUCCA6:GREEN FLUORESCENT PROTEIN fusion protein indicates YUCCA6 protein exhibits a nonplastidial subcellular localization in an unidentified intracellular compartment. Taken together, our results identify YUCCA6 as a functional member of the YUCCA family with unique roles in growth and development.
Auxin regulates a variety of physiological and developmental processes including cell elongation, cell division, cell differentiation, cell cycle, tropisms, and root and vascular development (Taiz and Zeiger, 2006). Although a number of auxin biosynthetic pathways have been identified their overlapping nature has prevented a clear elucidation of auxin biosynthesis. Independent groups reported Arabidopsis mutants with supernormal auxin phenotypes. These mutants exhibit hyperactivation of genes belonging to the YUCCA family encoding putative flavin monooxygenase enzymes that result in increased endogenous auxin levels. We isolated and characterized fertile dominant Arabidopsis thaliana hyt1-1D and hyt1-2D (yucca6-1D, -2D ) mutants that exhibit typical auxin overproduction phenotypic alterations such as epinastic cotyledons, increased apical dominance, and curled leaves. However, unlike other auxin overproduction mutants, yucca6 plants do not display short or hairy root phenotypes and lack morphological changes under dark conditions. In addition, yucca6-1D and yucca6-2D have extremely tall (over one meter) infloresences with extreme apical dominance and twisted cauline leaves. Microarray analyses revealed that expression of several IAA-inducible genes including Aux/IAA, SAUR, and GH3 are several-fold higher in yucca6 mutants than in wild type. Catalytic activity assays with recombinant YUCCA6 indicate that YUCCA6 is involved in a Trp-dependent auxin biosynthesis pathway. YUCCA6:GFP and YUCCA6:RFP fusion proteins indicate YUCCA6 protein exhibits a non-plastidial subcellular localization in an unidentified intracellular compartment. Based on these data, we conclude that Arabidopsis thaliana YUCCA6 as a member of the YUCCA family, functions in auxin biosynthesis. Furthermore, the unique phenotype of yucca6-1D, such as extremely delayed life span proposes possible unique roles of YUCCA6 in plant growth and development. Since YUCCA6 plays important roles in auxin biosynthesis, dramatically delayed senescence phenotype of yuc6-1D could be from elevated auxin levels in yuc6-1D. Next, I analyzed delayed senescence phenotypes in yuc6-1D. Although the first report for the involvement of auxin in controlling senescence was over fifty years ago, the role of auxin in plant senescence is still not clear. Both Arabidopsis thaliana yuc6-1D and 35S:YUC6 mutants that overexpress YUCCA6 contain elevated free IAA level compared to wild type plants and display dramatic delayed senescence phenotypes. Detached rosette leaves of YUCCA6 overexpressers exhibit delayed dark-induced senescence as well as delayed hormone-induced senescence. The monooxygenase enzyme activity of YUCCA6 is required for delaying senescence. Another hyper auxin mutant, Arabidosis thaliana yucca1-ox, also displays delayed dark-induced senescence as well as hormone-induced senescence. When excess free IAA from yuc6-1D was reduced, yuc6-1D leaves senesced normally implying that elevated free IAA retards leaf senescence in yuc6-1D. Free IAA measurements and auxin response gene expression profiles revealed that auxin homeostasis is actively down-regulated during senescence. The expression of GH3 family members involved in auxin amino acid conjugation was dramatically up-regulated during senescence, which suggests that plants tightly control auxin homeostasis during senescence possibly by conjugating free IAA. ANAC21/NAC1 and ANAC92/ATNAC6 expression is reduced in yuc6-1D during dark induced senescence compared to WT. Expression of miR164 in yuc6-1D is increased compared to WT whereas NAC1 expression is decreased in yuc6-1D. Our findings indicate that plants tightly control auxin levels which play critical roles in senescence, at least in part, through deactivation of auxin by conjugation.