Altering Tetrapyrrole Biosynthesis by Overexpressing Ferrochelatases (Fc1 and Fc2) Improves Photosynthetic Efficiency in Transgenic Barley
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agronomy Article Altering Tetrapyrrole Biosynthesis by Overexpressing Ferrochelatases (Fc1 and Fc2) Improves Photosynthetic Efficiency in Transgenic Barley Dilrukshi S. K. Nagahatenna 1, Jingwen Tiong 1, Everard J. Edwards 2 , Peter Langridge 1,3,* and Ryan Whitford 1 1 School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, PMB1, Glen Osmond, SA 5064, Australia; [email protected] (D.S.K.N.); [email protected] (J.T.); [email protected] (R.W.) 2 CSIRO Agriculture & Food, Locked Bag 2, Glen Osmond, SA 5064, Australia; [email protected] 3 Wheat Initiative, Julius-Kühn-Institute, Königin-Luise-Str 19, 14195 Berlin, Germany * Correspondence: [email protected]; Tel.: +61-883-137-171; Fax: +61-883-137-102 Received: 14 August 2020; Accepted: 7 September 2020; Published: 11 September 2020 Abstract: Ferrochelatase (FC) is the terminal enzyme of heme biosynthesis. In photosynthetic organisms studied so far, there is evidence for two FC isoforms, which are encoded by two genes (FC1 and FC2). Previous studies suggest that these two genes are required for the production of two physiologically distinct heme pools with only FC2-derived heme involved in photosynthesis. We characterised two FCs in barley (Hordeum vulgare L.). The two HvFC isoforms share a common catalytic domain, but HvFC2 additionally contains a C-terminal chlorophyll a/b binding (CAB) domain. Both HvFCs are highly expressed in photosynthetic tissues, with HvFC1 transcripts also being abundant in non-photosynthetic tissues. To determine whether these isoforms differentially affect photosynthesis, transgenic barley ectopically overexpressing HvFC1 and HvFC2 were generated and evaluated for photosynthetic performance. In each case, transgenics exhibited improved photosynthetic rate (Asat), stomatal conductance (gs) and carboxylation efficiency (CE), showing that both FC1 and FC2 play important roles in photosynthesis. Our finding that modified FC expression can improve photosynthesis up to ~13% under controlled growth conditions now requires further research to determine if this can be translated to improved yield performance under field conditions. Keywords: tetrapyrrole; ferrochelatase; heme; chlorophyll; photosynthesis 1. Introduction Production of the major cereal crops needs to improve to feed future food demands driven by population growth. This task will be challenged by production constraints due to increased climatic variability. Improving photosynthetic performance of rain-fed cereals may be a step towards achieving higher crop yields on limited arable land. As photosynthesis is a highly complex and regulated physiological process, the identification of genes and processes capable of enhancing photosynthetic efficiency is a high priority [1–3]. Knowledge of these genes and processes will allow researchers and plant breeders to identify, track and ultimately deploy improved photosynthetic traits. Tetrapyrroles are key components of photosynthesis. All higher plants synthesise two major tetrapyrroles, chlorophyll and heme [4]. In plastids, chlorophyll plays a vital role in the capture and conversion of light energy for photosynthesis [5], whilst heme is an integral component of the photosynthetic cytochrome b6f complex, necessary for photosynthetic electron transport [6,7]. Unlike chlorophyll, heme has a wide distribution within the cell and is required for a number of other cellular functions. For instance, in both the mitochondria and endoplasmic reticulum, heme is involved Agronomy 2020, 10, 1370; doi:10.3390/agronomy10091370 www.mdpi.com/journal/agronomy Agronomy 2020, 10, x FOR PEER REVIEW 2 of 15 photosynthetic cytochrome b6f complex, necessary for photosynthetic electron transport [6,7]. Unlike chlorophyll, heme has a wide distribution within the cell and is required for a number of other Agronomycellular2020 functions., 10, 1370 For instance, in both the mitochondria and endoplasmic reticulum, heme2 of 15 is involved in electron transport through respiratory cytochromes, cytochrome b5 and P450s. In peroxisomes, it acts as a co-factor for activating enzymes which detoxify reactive oxygen species in(ROS) electron such transport as, catalase through and ascorbate respiratory peroxidase cytochromes, [8]. Recently, cytochrome it was b5 proposed and P450s. that In heme peroxisomes, serves as a itplastid acts as signal a co-factor for modulating for activating expression enzymes of which a number detoxify of chloroplast reactive oxygen biogenesis-associated species (ROS) such nuclear as, catalasegenes (retrograde and ascorbate signalling) peroxidase [9–13]. [8]. Studies Recently, to date it was show proposed that tetrapyrrole that heme servesbiosynthesis as a plastid is modulated signal forat modulatingtwo strict control expression points: of a numberaminolevulinic of chloroplast acid synthesis biogenesis-associated [14] and at the nuclear branch genes point (retrograde between signalling)chlorophyll [9 –and13]. heme Studies synthesis to date (Figure show that1) [4,15–17]. tetrapyrrole At the biosynthesis branch point, is modulatedProtoporphyrin at two IX strict(Proto controlIX) serves points: as the aminolevulinic common substrate acid synthesis for tetrapyrrole [14] and biosynthesis. at the branch Insertion point between of Mg2+ chlorophyll into Proto IX and by hemeMg-chelatase synthesis forms (Figure chlorophyll,1)[ 4,15–17 ].whereas At the branch insertion point, of Fe Protoporphyrin2+ by Ferrochelatase IX (Proto (FC) IX) is servesnecessary as the for 2+ commonheme production substrate for [18]. tetrapyrrole biosynthesis. Insertion of Mg into Proto IX by Mg-chelatase forms chlorophyll, whereas insertion of Fe2+ by Ferrochelatase (FC) is necessary for heme production [18]. Figure 1. The tetrapyrrole biosynthesis pathway with major end products (bold text) and some catalytic enzymes.Figure 1. GluTR; The tetrapyrrole Glutamyl-tRNA biosynthesis reductase, pathway Protogen with IX oxidase;major end Protoporphyrinogen products (bold text) IX oxidase. and some catalytic enzymes. GluTR; Glutamyl-tRNA reductase, Protogen IX oxidase; Protoporphyrinogen IX In plants studied so far, there is evidence for two FC isoforms, which are each encoded by a single oxidase. gene (FC1 and FC2). Both FC isoforms exist as 36–42 kDa monomers [19], and are inferred to have similarIn catalytic plants studied properties, so far, substrate there is affi evidencenity and fo specificity,r two FC basedisoforms, on in which vitro assaysare each [20 encoded]. However, by a thesingle two FCgenes have (FC1 distinct and FC2 expression). Both FC profiles. isoformsFC1 existis abundantly as 36–42 kDa expressed monomers in all plant[19], and tissues, are includinginferred to roots,have whereassimilar catalyticFC2 transcript properties, levels substrate are found affinity only in and aerial specificity, plant parts based [19, 21on– 24in]. vitroIn vitro assaysimport [20]. assaysHowever, indicate the thattwo both FCs FC1haveand distinctFC2 are expression localized toprofiles. the stroma, FC1 is thylakoid abundantly and envelopeexpressed membranes in all plant oftissues, the chloroplast including [roots,20,25, 26whereas], while FC2FC1 transcriptis additionally levels are imported found only into in mitochondria aerial plant parts [21, 25[19,21–24].,27–29]. TheseIn vitro diff erencesimport assays have led indicate to the that proposition both FC1 that and each FC2 FC are has localized a distinct to rolethe stroma, in plant thylakoid metabolism. and Dualenvelope targeting membranes of FC1 to bothof the chloroplasts chloroplast and [20,25,26], mitochondria while has beenFC1 disputedis additionally in subsequent imported studies. into Formitochondria example, Lister [21,25,27–29]. et al. [30 These] were di unablefferences to have detect ledFC1 to thein Arabidopsis propositionmitochondria, that each FC has whilst a distinct pea mitochondria,role in plant inmetabolism. which previous Dual importtargeting assays of FC1 had to been both conducted, chloroplasts appeared and mitochondria to accept a variety has been of chloroplast-specificdisputed in subsequent proteins studie in additions. For example, to Arabidopsis Lister et FC1 al. [[30]30]. were Masuda unable et al. to [ 31detect] also FC1 found in Arabidopsis that FC1 andmitochondria,FC2 in cucumber whilst are pea both mitochondria, solely targeted in towhich chloroplasts. previous import assays had been conducted, appearedFC2, but to notacceptFC1, ahas variety recently of beenchloroplast-specific demonstrated toproteins positively in co-expressaddition to with Arabidopsis light-responsive FC1 [30]. photosyntheticMasuda et al. genes[31] also [23 ].foundArabidopsis that FC1 fc2 andknock-down FC2 in cucumber mutants are (fc2–1 both) exhibited solely targeted a significant to chloroplasts. reduction in plastidFC2 cytochromes,, but not FC1, has cytochrome recently b6f-boundbeen demonstrated heme and to anpositively impairment co-express of photosynthetic with light-responsive electron transportphotosynthetic and photosystem genes [23]. II (PSII)Arabidopsis efficiency fc2 [knock-down23,32]. In comparison, mutants Arabidopsis(fc2–1) exhibited fc1–1 knock-down a significant mutantsreduction did innot plastid display cytochromes, obvious defectscytochrome in photosynthetic b6f-bound heme development and an impairment but exhibited of photosynthetic reduction inelectron cytochromes transport in microsomal and photosystem fraction II (PSI andI) peroxidase efficiency [23,32]. activity, In suggesting comparison,