Lawrence Berkeley National Laboratory Recent Work Title Transcript, protein and metabolite temporal dynamics in the CAM plant Agave. Permalink https://escholarship.org/uc/item/2p45c5cx Journal Nature plants, 2(12) ISSN 2055-0278 Authors Abraham, Paul E Yin, Hengfu Borland, Anne M et al. Publication Date 2016-11-21 DOI 10.1038/nplants.2016.178 Peer reviewed eScholarship.org Powered by the California Digital Library University of California ARTICLES PUBLISHED: 21 NOVEMBER 2016 | ARTICLE NUMBER: 16178 | DOI: 10.1038/NPLANTS.2016.178 Transcript, protein and metabolite temporal dynamics in the CAM plant Agave Paul E. Abraham1,HengfuYin2,AnneM.Borland2,3, Deborah Weighill2,4,SungDonLim5, Henrique Cestari De Paoli2, Nancy Engle2,PietC.Jones2,4,RyanAgh2, David J. Weston2, Stan D. Wullschleger6, Timothy Tschaplinski2, Daniel Jacobson2,4, John C. Cushman5, Robert L. Hettich1, Gerald A. Tuskan2 and Xiaohan Yang2* Already a proven mechanism for drought resilience, crassulacean acid metabolism (CAM) is a specialized type of fi photosynthesis that maximizes water-use ef ciency by means of an inverse (compared to C3 and C4 photosynthesis) day/ night pattern of stomatal closure/opening to shift CO2 uptake to the night, when evapotranspiration rates are low. A systems-level understanding of temporal molecular and metabolic controls is needed to define the cellular behaviour underpinning CAM. Here, we report high-resolution temporal behaviours of transcript, protein and metabolite abundances across a CAM diel cycle and, where applicable, compare the observations to the well-established C3 model plant Arabidopsis. A mechanistic finding that emerged is that CAM operates with a diel redox poise that is shifted relative to that in Arabidopsis. Moreover, we identify widespread rescheduled expression of genes associated with signal transduction mechanisms that regulate stomatal opening/closing. Controlled production and degradation of transcripts and proteins represents a timing mechanism by which to regulate cellular function, yet knowledge of how this molecular timekeeping regulates CAM is unknown. Here, we provide new insights into complex post-transcriptional and -translational hierarchies that govern CAM in Agave. These data sets provide a resource to inform efforts to engineer more efficient CAM traits into economically valuable C3 crops. fi he water-use ef ciencyof Agave spp. hinges on crassulacean acid opening/closing of stomata that distinguish CAM from C3 photosyn- metabolism (CAM), a specialized mode of photosynthesis that thesis imply that the bioengineering of CAM will require a temporal Tevolved from ancestral C3 photosynthesis in response to water reprogramming of metabolism in the C3 host. Therefore, key chal- 1 ∼ and CO2 limitation and is found in 6.5% of higher plants. lenges for CAM biodesign will be to establish how many genes Whereas C3 photosynthesis produces a three-carbon (3-C) molecule must be reprogrammed in a diel manner to modify the behaviour fi for carbon xation during the day, CAM generates a four-carbon of C3 plants to perform CAM and to identify which functional or organic acid from carbon fixation at night. In CAM, this nocturnal car- mechanistic governing principles are shared among the diel tran- boxylation reaction is catalysed by phosphoenolpyruvate carboxylase scriptional and translational dynamics of C3 and CAM. (PEPC), and the 3-C substrate phosphoenolpyruvate (PEP) is supplied Generating an integrated functional -omics dataset (transcrip- by the glycolytic breakdown of carbohydrate formed during the pre- tomics, proteomics and metabolomics) for a CAM species is an vious day. The nocturnally accumulated malic acid is stored overnight essential first step for providing global insight into the complete in a central vacuole, and during the subsequent day malate is decar- set of genes controlling the metabolic steps of CAM, for revealing boxylated to release CO2 at an elevated concentration for Rubisco in genes in co-occurrence networks, and for determining the func- the chloroplast. The diel separation of carboxylases in CAM is tional consequences of diel co-regulation of transcription and trans- fi accompanied by an inverse (compared with C3 and C4 photosyn- lation. In the present study, temporal pro les of the transcriptome, thesis-performing species) day/night pattern of stomatal closure/ proteome and metabolome of CAM-performing leaves from the fi fi opening that results in improved water-use ef ciency (CO2 xed per obligate CAM species Agave americana were investigated across a unit water lost) that is up to sixfold higher than that of C3 photosyn- 12 h/12 h light/dark diel cycle. With this experimental design, we fi thesis plants and up to threefold higher than that of C4 photosynthesis sought to: (1) identify temporally de ned clusters of co-regulated plants under comparable conditions2. genes, (2) define diel shifts in gene expression between CAM- and fi The frequent emergence of CAM from C3 photosynthesis through- C3-speci c gene networks and (3) describe the temporal dynamics out evolutionary history implies that all of the enzymes required for between gene expression profiles and protein abundance profiles 1,3 CAM are homologues of ancestral forms found in C3 species .As across the 24 hour light/dark CAM cycle. such, the CAM pathway has been identified as a target for synthetic biology because it offers the potential to engineer improved water- Results fi 4–6 use ef ciency in C3 crops . However, the day/night separation of car- Metabolic reprogramming in CAM-performing leaves. CAM fi boxylation and decarboxylation processes and the inverse night/day plants are classi ed according to the amount of atmospheric CO2 1Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA. 2Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA. 3 School of Biology, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK. 4The Bredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee, Knoxville, Tennessee 37996, USA. 5Department of Biochemistry and Molecular Biology, University of Nevada, MS330, Reno, Nevada 89557-0330, USA. 6Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA. *e-mail: [email protected] NATURE PLANTS | www.nature.com/natureplants 1 © 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. ARTICLES NATURE PLANTS DOI: 10.1038/NPLANTS.2016.178 that is taken up at night1,7,8.ForA. americana, the magnitude of include glycolysis, TCA cycle, OPP, nitrogen assimilation and nocturnal net CO2 uptake changes according to leaf age, with a respiratory electron transport. progressive shift from predominantly C3 photosynthesis in the youngest leaf to increasing CAM activity with leaf age (Fig. 1a; Temporal dynamics of gene expression across a CAM diel series. Supplementary Table 1). We limited all sampling for metabolites, Using the same leaf material as sampled for the metabolite profiles, transcriptome and proteome to the fourth fully expanded leaf, RNA sequencing (RNA-Seq) was performed across eight time which is a mature CAM-performing leaf under the controlled points at 3 hour intervals in biological triplicates. RNA-Seq- environmental conditions used here (day/night temperature 25/15 °C; derived transcript profiles were obtained, and the total abundance − − 12 hour photoperiod, photon flux density 540 µmol m 2 s 1 at of each transcript was assessed after normalizing the number of plant height). reads per kilobase and normalizing per million reads (RPKM). In We inspected gas chromatography mass spectrometry (MS) pro- total, 47,499 transcripts were observed in mature leaves of Agave. files of 64 abundant metabolites in the Agave diel cycle (Fig. 1b; For quantitative analyses, an empirically derived threshold Supplementary Table 2), and then compared a subset of these to (maximum RPKM ≥ 5.02 and minimum average RPKM ≥ 3.483; those in an Arabidopsis C3 leaf (Supplementary Table 3; Fig. 1c). Supplementary Note 5) was applied to remove low-abundance Unlike C3 leaves, in which malic and fumaric acid levels increase transcripts that had large variance across the entire transcriptomic during the day and decrease during the night9,10, Agave leaves data set24 resulting in 37,808 transcripts (Supplementary Table 4). accumulate malic acid at night, a defining feature of the nocturnal Examination of the data revealed that 82% (31,126) of transcripts fi CO2 xation that occurs during CAM. Agave leaves also accumu- were expressed throughout the entire 24 hour period. Pearson lated fumaric acid during the night, which is consistent with the correlations were computed and high reproducibility was found at relatively high night-time fluxes of carbon passing through the an average Pearson correlation coefficient of 0.91. tricarboxylic acid (TCA) cycle and the carbon exchange between On the basis of paired t-tests, the expression patterns of 21,168 malate and fumarate11 reported for CAM plants. The diel abun- transcripts that showed at least a twofold change from their mean dance profile of sucrose, which is reciprocal to that of malic acid, value with P < 0.05 between one or more time points across the diel provides support for the hypothesis that Agave uses soluble cycle (Supplementary Table 5) were grouped into nine major clusters sugars, mainly fructans, oligofructans, fructose, glucose and based on similarity of expression patterns identified using the sucrose, as potential carbon sources