Metabolite Profiles of Maize Leaves in Drought,Heat, and Combined Stress Field Trials Reveal the Relationship between Metabolism and Grain Yield

The development of abiotic stress-resistant cultivars is of premium importance for the agriculture of developing countries. Further progress in maize (Zea mays) performance under stresses is expected by combining marker-assisted breeding with metabolite markers. In order to dissect metabolic responses and to identify promising metabolite marker candidates, metabolite profiles of maize leaves were analyzed and compared with grain yield in field trials. Plants were grown under well-watered conditions (control) or exposed to drought, heat, and both stresses simultaneously. Trials were conducted in 2010 and 2011 using 10 tropical hybrids selected to exhibit diverse abiotic stress tolerance. Drought stress evoked the accumulation of many amino acids, including isoleucine, valine, threonine, and 4-aminobutanoate, which has been commonly reported in both field and greenhouse experiments in many plant species. Two photorespiratory amino acids, glycine and serine, and myoinositol also accumulated under drought. The combination of drought and heat evoked relatively few specific responses, and most of the metabolic changes were predictable from the sum of the responses to individual stresses. Statistical analysis revealed significant correlation between levels of glycine and myoinositol and grain yield under drought. Levels of myoinositol in control conditions were also related to grain yield under drought. Furthermore, multiple linear regression models very well explained the variation of grain yield via the combination of several metabolites. These results indicate the importance of photorespiration and raffinose family oligosaccharide metabolism in grain yield under drought and suggest single or multiple metabolites as potential metabolic markers for the breeding of abiotic stress-tolerant maize.The increasing world population coupled to environmental deterioration is creating ever greater pressure on our capacity for sustainable food productivity. Alongside biotic stresses, abiotic stresses such as drought, heat, salinity, and nutrient deficiency greatly reduce yields in crop fields either when present alone or in combination. Breeding for more resilient crops, therefore, is one of the major approaches to cope with the increasing challenges in world agriculture. Considerable research effort has thus been invested in order to dissect plant responses to individual stresses at various levels (for review, see Urano et al., 2010; Lopes et al., 2011; Obata and Fernie, 2012), but the interaction between different stresses has been far less investigated (Cairns et al., 2012b, 2013; Suzuki et al., 2014). In general, the combination of stresses additively affects plant physiology (i.e. the symptoms of the individual stresses appear simultaneously) and synergistically diminishes the yield and productivity of plants (Keleş and Öncel, 2002; Giraud et al., 2008; Vile et al., 2012; Suzuki et al., 2014). The molecular responses, however, are not simply additive and are rarely predicted from the responses to individual stresses (Rizhsky et al., 2002, 2004; Prasch and Sonnewald, 2013; Rasmussen et al., 2013). Information from carefully controlled greenhouse experiments has begun to dissect the molecular mechanisms by which plants, in particular Arabidopsis (Arabidopsis thaliana), respond to drought and temperature stresses (Skirycz et al., 2010, 2011; Skirycz and Inzé, 2010; Bowne et al., 2012; Tardieu, 2012; Verkest et al., 2015). Our knowledge of the molecular basis of the responses of crop species in a field environment, however, is considerably less well advanced (Araus et al., 2008; Cabrera-Bosquet et al., 2012). That said, a large number of genotypes have been generated on the basis of their resistance to both biotic and abiotic stresses (for review, see Bänziger et al., 2006; Takeda and Matsuoka, 2008; Cooper et al., 2014), and the genome sequencing and molecular characterization of a range of stress-tolerant plant species have recently been reported (Wu et al., 2012; Ma et al., 2013; Bolger et al., 2014; Tohge et al., 2014). These studies are not only important as basic research for further studies in crops but also are a prerequisite in the development of molecular marker-based approaches to improve crop tolerance to stress.As a first step toward this goal, a deeper understanding of the plant responses to the stressful environment, especially those to multiple stress conditions under field conditions, is crucial for the improvement of stress-tolerant crops. This is important on two levels: (1) in the field, singular abiotic stresses are rare; and (2) yield and stress adaptation are complex traits that render breeding gains slower than would be expected under optimal conditions (Bruce et al., 2002). Recent studies have revealed that the response of plants to combinations of two or more stress conditions is unique and cannot be directly extrapolated from their responses to the different stresses when applied individually. This would be a result of complex combinations of different, and sometimes opposing, responses in signaling pathways, including those that may interact and inhibit one another (Prasch and Sonnewald, 2013; Rasmussen et al., 2013; Suzuki et al., 2014).Maize (Zea mays) is grown in over 170 million ha worldwide, of which 130 million ha are in less-developed countries (FAO, 2014). In sub-Saharan Africa, maize is a staple crop; however, yields in this region have stagnated at less than 2 tons ha⁻¹, while maize yields worldwide have continued to increase (Cairns et al., 2012a). Low yields in sub-Saharan Africa are largely associated with drought stress (DS) and low soil fertility (Bänziger and Araus, 2007). Additionally, simulation studies indicate that maize yield in Africa is likely to be significantly impaired by heat stress (HS; Lobell and Burke, 2010; Lobell et al., 2011), such as can be anticipated as a result of the changes in climate predicted for the coming decades (Müller et al., 2011). Moreover, the sensitivity of maize yield to heat is exacerbated under drought conditions (Lobell et al., 2011; Cairns et al., 2012a, 2012b, 2013). Therefore, the development of maize germplasm tolerant to drought and heat conditions is of utmost importance to both increase yields and offset predicted yield losses under projected climate change scenarios (Easterling et al., 2007), especially in sub-Saharan Africa. While direct selection for grain yield under DS has resulted in admirable gains in grain yield under stress (Bänziger et al., 2006; Cairns et al., 2013), further improvement requires the incorporation of additional selection traits (Cairns et al., 2012a, 2012b). In recent years, genetic and phenotypic markers have been searched extensively for drought tolerance of maize by high-throughput genomic and phenotyping approaches, respectively (Tuberosa and Salvi, 2006; Wen et al., 2011; Araus et al., 2012; Cairns et al., 2013; Prasanna et al., 2013; Araus and Cairns, 2014; Tsonev et al., 2014). Moreover, metabolic markers started to draw attention due to their close relationship with yield phenotypes (Fernie and Schauer, 2009; Redestig et al., 2011; Riedelsheimer et al., 2012a, 2012b; Witt et al., 2012; Degenkolbe et al., 2013). The accumulation of some metabolites has been reported to be directly related to the performance of potato (Solanum tuberosum) cultivars in beetle resistance in the field (Tai et al., 2014). Additionally, identical genomic regions were mapped as both agronomic and metabolic quantitative trait loci in field-grown maize and wheat (Triticum aestivum), indicating the utility of metabolic traits for breeding selection (Riedelsheimer et al., 2012b; Hill et al., 2015). A recent study showed that genetic gains in maize grain yield under DS were higher using a molecular marker-based approach than conventional breeding (Beyene et al., 2015).Here, we focused on the relationship between leaf metabolites and grain yield under drought, heat, and simultaneous drought and heat conditions in the field. The negative effect of DS on maize yield is especially acute during the reproductive stage between tassel emergence and early grain filling (Grant et al., 1989), when it is believed to induce premature seed desiccation and to limit grain filling. Grain is more susceptible to DS than vegetative tissues; therefore, the prediction of grain yield from the physiological parameter of leaves is a challenge (Sangoi and Salvador, 1998; Khodarahmpour and Hamidi, 2011). Nevertheless, maize yield is dependent on both the assimilate supply to the kernel (source) and the potential of the kernel to accommodate this assimilate (sink potential; Jones and Simmons, 1983). Breeding for modern temperate hybrids has focused more on the sink potential, particularly under stress conditions (Tollenaar and Lee, 2006); therefore, there should be considerable potential remaining to improve source ability. DS and HS would be anticipated largely to affect leaf metabolism and especially photosynthesis, compromising the source capacity of leaves (Chaves et al., 2009; Lawlor and Tezara, 2009; Osakabe et al., 2014). In keeping with this, drought was found to have the most dramatic effect on the metabolite composition in leaves compared with other organs in our previous greenhouse experiments (Witt et al., 2012). Since the source ability is closely related to leaf metabolism, the leaf metabolite profile should have a close relationship to grain yield particularly under conditions of stress. Given that several recent studies have indicated the importance of metabolic preadaptation to various stress tolerances in plants (Sanchez et al., 2011; Benina et al., 2013), we also postulate that basal metabolite levels under optimal growth conditions could be correlated to stress tolerance. In order to test this, metabolite profiles of the leaf blades of 10 hybrids were analyzed in field experiments conducted at the International Maize and Wheat Improvement Center (CIMMYT) subtropical experimental station in 2010 and 2011 in which the plants were exposed to singular or combined drought and heat stresses (DS+HS; Cairns et al., 2012a, 2013). The results are discussed both in the context of current models of stress tolerance and with respect to their practical implications for future breeding strategies.     

Enhanced Photosynthesis and Growth in atquac1 Knockout Mutants Are Due to Altered Organic Acid Accumulation and an Increase in Both Stomatal and Mesophyll Conductance

Stomata control the exchange of CO₂ and water vapor in land plants. Thus, whereas a constant supply of CO₂ is required to maintain adequate rates of photosynthesis, the accompanying water losses must be tightly regulated to prevent dehydration and undesired metabolic changes. Accordingly, the uptake or release of ions and metabolites from guard cells is necessary to achieve normal stomatal function. The AtQUAC1, an R-type anion channel responsible for the release of malate from guard cells, is essential for efficient stomatal closure. Here, we demonstrate that mutant plants lacking AtQUAC1 accumulated higher levels of malate and fumarate. These mutant plants not only display slower stomatal closure in response to increased CO₂ concentration and dark but are also characterized by improved mesophyll conductance. These responses were accompanied by increases in both photosynthesis and respiration rates, without affecting the activity of photosynthetic and respiratory enzymes and the expression of other transporter genes in guard cells, which ultimately led to improved growth. Collectively, our results highlight that the transport of organic acids plays a key role in plant cell metabolism and demonstrate that AtQUAC1 reduce diffusive limitations to photosynthesis, which, at least partially, explain the observed increments in growth under well-watered conditions.Stomata are functionally specialized microscopic pores that control the essential exchange of CO₂ and H₂O with the environment in land plants. Stomata are found on the surfaces of the majority of the aerial parts of plants, rendering them as the main control point regulating the flow of gases between plants and their surrounding atmosphere. Accordingly, the majority of water loss from plants occurs through stomatal pores, allowing plant transpiration and CO₂ absorption for the photosynthetic process (Bergmann and Sack, 2007; Kim et al., 2010). The maintenance of an adequate water balance through stomatal control is crucial to plants because cell expansion and growth require tissues to remain turgid (Sablowski and Carnier Dornelas, 2014), and minor reductions in cell water volume and turgor pressure will therefore compromise both processes (Thompson, 2005). As a result, the high sensitivity of plant tissues to turgor has prompted the use of reverse genetic studies in attempt to engineer plants with improved performance (Cowan and Troughton, 1971; Xiong et al., 2009; Borland et al., 2014; Franks et al., 2015).In most land plants, not only redox signals invoked by shifts in light quality (Busch, 2014) but also the transport of inorganic ions (e.g. K⁺, Cl⁻, and NO⁻₃) as well as metabolites such as the phytohormone abscisic acid (ABA), Suc, and malate, are important players controlling stomatal movements (Hetherington, 2001; Roelfsema and Hedrich, 2005; Pandey et al., 2007; Blatt et al., 2014; Kollist et al., 2014). In this context, although organic acids in plants is known to support numerous and diverse functions both within and beyond cellular metabolism, only recently have we obtained genetic evidence to support that modulation of guard cell malate and fumarate concentration can greatly influence stomatal movements (Nunes-Nesi et al., 2007; Araújo et al., 2011b; Penfield et al., 2012; Medeiros et al., 2015). Notably malate, in particular, has been considered as a key metabolite and one of the most important organic metabolites involved in guard cell movements (Hedrich and Marten, 1993; Fernie and Martinoia, 2009; Meyer et al., 2010). During stomatal aperture, the flux of malate into guard cells coupled with hexoses generated on starch breakdown lead to decreases in the water potential, and consequently, water uptake by the guard cells ultimately opens the stomata pore (Roelfsema and Hedrich, 2005; Vavasseur and Raghavendra, 2005; Lee et al., 2008). On the other hand, during stomatal closure, malate is believed to be converted into starch, which has no osmotic activity (Penfield et al., 2012) or, alternatively, is released from the guard cells to the surrounding apoplastic space (Lee et al., 2008; Negi et al., 2008; Vahisalu et al., 2008; Meyer et al., 2010).The role of organic acids on the stomatal movements has been largely demonstrated by studies related to malate transport (Lee et al., 2008; Meyer et al., 2010; Sasaki et al., 2010). In the last decade, two protein families were identified and functionally characterized to be directly involved with organic acid transport at the guard cell plasma membrane and to be required for stomatal functioning (Lee et al., 2008; Meyer et al., 2010; Sasaki et al., 2010). In summary, AtABCB14, a member of the ABC (ATP binding cassette) family, which is involved in malate transport from apoplast to guard cells, was described as a negative modulator of stomatal closure induced by high CO₂ concentration; notably, exogenous application of malate minimizes this response (Lee et al., 2008). In addition, members of a small gene family, which encode the anion channels SLAC1 (slow anion channel 1) and four SLAC1-homologs (SLAHs) in Arabidopsis (Arabidopsis thaliana), have been described to be involved in stomatal movements. SLAC1 is a well-documented S-type anion channel that preferentially transports chloride and nitrate as opposed to malate (Vahisalu et al., 2008, 2010; Geiger et al., 2010; Du et al., 2011; Brandt et al., 2012; Kusumi et al., 2012). Lack of SLAC1 in Arabidopsis and rice (Oryza sativa) culminated in a failure in stomatal closure in response to high CO₂ levels, low relative humidity, and dark conditions (Negi et al., 2008; Vahisalu et al., 2008; Kusumi et al., 2012). Although mutations in AtSLAC1 impair S-type anion channel functions as a whole, the R-type anion channel remained functional (Vahisalu et al., 2008). Indeed, a member of the aluminum-activated malate transporter (ALMT) family, AtALMT12, an R-type anion channel, has been demonstrated to be involved in malate transport, particularly at the plasma membrane of guard cells (Meyer et al., 2010; Sasaki et al., 2010). Although AtALMT12 is a member of ALMT family, it is not activated by aluminum, and therefore Meyer et al. (2010) proposed to rename it as AtQUAC1 (quick-activating anion channel 1; Imes et al., 2013; Mumm et al., 2013). Hereafter, we will follow this nomenclature. Deficiency of a functional AtQUAC1 has been documented to lead to changes in stomatal closure in response to high levels of CO₂, dark, and ABA (Meyer et al., 2010). Taken together, these studies have clearly demonstrated that both S- and R-type anion channels are key modulators of stomatal movements in response to several environmental factors.Despite a vast number of studies involving the above-mentioned anion channels, little information concerning the metabolic changes caused by their impairment is currently available. Such information is important to understand stomatal movements, mainly considering that organic acids, especially the levels of malate in apoplastic/mesophyll cells, have been highlighted as of key importance in leaf metabolism (Fernie and Martinoia, 2009; Araújo et al., 2011a, 2011b; Lawson et al., 2014; Medeiros et al., 2015). Here, we demonstrate that a disruption in the expression of AtQUAC1, which leads to impaired stomatal closure (Meyer et al., 2010), was accompanied by increases in mesophyll conductance (g_m), which is defined as the conductance for the transfer of CO₂ from the intercellular airspaces (C_i) to the sites of carboxylation in the chloroplastic stroma (C_c). By further characterization of atquac1 knockout plants, we demonstrated that reduced diffusive limitations resulted in higher photosynthetic rates and altered respiration that, in turn, led to enhanced biomass accumulation. Overall, the results obtained are discussed both in terms of the importance of organic acid transport in plant cell metabolism and with regard to the contribution that it plays in the regulation of both stomatal function and growth.     

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.     

Mechanical Failure of Fine Root Cortical Cells Initiates Plant Hydraulic Decline during Drought

Root systems perform the crucial task of absorbing water from the soil to meet the demands of a transpiring canopy. Roots are thought to operate like electrical fuses, which break when carrying an excessive load under conditions of drought stress. Yet the exact site and sequence of this dysfunction in roots remain elusive. Using in vivo x-ray computed microtomography, we found that drought-induced mechanical failure (i.e. lacunae formation) in fine root cortical cells is the initial and primary driver of reduced fine root hydraulic conductivity (Lp_r) under mild to moderate drought stress. Cortical lacunae started forming under mild drought stress (−0.6 MPa Ψ_stem), coincided with a dramatic reduction in Lp_r, and preceded root shrinkage or significant xylem embolism. Only under increased drought stress was embolism formation observed in the root xylem, and it appeared first in the fine roots (50% loss of hydraulic conductivity [P₅₀] reached at −1.8 MPa) and then in older, coarse roots (P₅₀ = −3.5 MPa). These results suggest that cortical cells in fine roots function like hydraulic fuses that decouple plants from drying soil, thus preserving the hydraulic integrity of the plant’s vascular system under early stages of drought stress. Cortical lacunae formation led to permanent structural damage of the root cortex and nonrecoverable Lp_r, pointing to a role in fine root mortality and turnover under drought stress.Root systems of woody plants consist of both coarse and nonwoody fine roots. Fine roots can constitute as little as 1% of the total root surface area (Kramer and Bullock, 1966), yet are critically important for biogeochemical cycling in terrestrial ecosystems as they constitute the primary exchange surface between plants and soil (Jackson et al., 1997). They are responsible for the vast majority of water absorption in woody root systems (Gambetta et al., 2013; Kramer and Boyer, 1995; Kramer and Bullock, 1966) and mediate backward flow of water from a plant to the soil via a process called hydraulic redistribution, which can alter regional climate (Richards and Caldwell, 1987; Lee et al., 2005). Fine roots also modify the soil through carbon exudation and stimulation of microbial activity (McCormack et al., 2015), and their production and annual turnover represent 33% of global net primary productivity (Jackson et al., 1997; McCormack et al., 2015). Elucidating details of fine root function and responses to stress can thus improve our understanding of how these plant organs can influence ecosystem carbon, nutrient, and water cycles.Fine roots are traditionally defined as all roots <2-mm diameter, but recent work has emphasized the need to delineate this diameter class into distinct functional groups. By separating fine roots into a shorter-lived absorptive pool and a longer-lived transport pool, McCormack et al. (2015) showed that fine root functionality can alter estimates of global net primary productivity by 30%. This work highlights our still-limited understanding of fine root functionality, the mechanisms underlying their lifespan and turnover, and how those traits respond to abiotic stress (Lukac, 2012; Tierney and Fahey, 2002; Guo et al., 2008). Fine root mortality during drought has been linked to increased root respiration and inhibited photosynthate transport to roots (e.g. Marshall, 1986) but could also be attributed to hydraulic dysfunction (Jackson et al., 2000). Portions of root systems are thought to operate analogously to a hydraulic fuse in an electrical circuit and designed to fail hydraulically when carrying excessive current under drought stress (Zimmermann, 1983; Jackson et al., 2000). However, the exact location of these hydraulic fuses in the root system has yet to be identified. Axial water transport in the xylem is considered a weak link, as roots of numerous species have been shown to be more susceptible to drought-induced xylem embolism compared to other organs within the same plant (i.e. trunks, stems, tap roots; Alder et al., 1996; Hacke and Sauter, 1996; McElrone et al., 2004; Pratt et al., 2015; Johnson et al., 2016). Moreover, Sperry and Ikeda (1997) found that smaller roots were the most vulnerable plant organ to xylem embolism, which would localize failure to inexpensive, distal, and easily replaceable portions of a root system. Such a design is considered effective, because it is widely assumed that the hydraulic capacity of smaller distal roots is readily repaired upon rewatering via xylem embolism removal (Domec et al., 2006; Jackson et al., 2000). While much work has demonstrated that xylem embolism reduces hydraulic capacity under severe drought stress (Brodribb et al., 2016a, 2016b; Choat et al., 2012), its contribution under mild to moderate stress is less clear (Choat et al., 2016; Cochard and Delzon, 2013; Cochard et al., 2013; McElrone et al., 2012; Wheeler et al., 2013; Choat et al., 2010; Torres-Ruiz et al., 2015). Work is still needed to resolve the location and sequence of root hydraulic dysfunction under drought and what tissues are involved in each stage of this process, especially under mild stress where fine root hydraulic conductivity (Lp_r) is known to decrease dramatically (Aroca et al., 2012).Before entering the xylem for long distance transport, water absorbed by roots must traverse a series of cell layers that include the epidermis, cortex, and endodermis (Steudle and Peterson 1998). Hydraulic resistance is much greater along this radial pathway compared to the axial transport pathway in the xylem (e.g. Frensch and Hsiao, 1993; Frensch and Steudle, 1989). The resistance differential between radial and axial pathways persists or increases in magnitude as fine Lp_r decreases under mild to moderate drought stress. While decreased fine root permeability under drought has been attributed to root shrinkage (Passioura, 1988; Nobel and Cui, 1992), changes in membrane permeability via aquaporins (Maurel et al., 2015; Aroca et al., 2012; North, 2004), development of suberized apoplastic barriers over longer periods of drought (Barrios-Masias et al., 2015; North and Nobel, 1991), or mechanical damage in cortical cells (i.e. lacunae formation; North and Nobel, 1991), the integration of these responses particularly under mild stress is still lacking. Elucidating the physiological mechanism that drives this response could help to resolve long-standing questions about fine root functionality, lifespan, and turnover.Here, we originally aimed to study whether fine roots function as the primary hydraulic fuse that disconnects a plant from drying soil. We studied the sequence of events during soil drying from saturated to severe drought conditions in coarse and fine roots of grapevines, which are considered a model species and have long been characterized as highly susceptible to drought-induced embolism. While performing these experiments, we discovered that the fine root cortex was radically changing under mild drought stress that preceded any embolism formation. We then performed hydraulic measurements and fluorescence light microscopy to investigate how fine Lp_r is affected by the formation of cortical lacunae that should significantly increase radial hydraulic resistance to flow.     

Mitochondrial Alternative Oxidase Maintains Respiration and Preserves Photosynthetic Capacity during Moderate Drought in Nicotiana tabacum

The mitochondrial electron transport chain includes an alternative oxidase (AOX) that is hypothesized to aid photosynthetic metabolism, perhaps by acting as an additional electron sink for photogenerated reductant or by dampening the generation of reactive oxygen species. Gas exchange, chlorophyll fluorescence, photosystem I (PSI) absorbance, and biochemical and protein analyses were used to compare respiration and photosynthesis of Nicotiana tabacum ‘Petit Havana SR1’ wild-type plants with that of transgenic AOX knockdown (RNA interference) and overexpression lines, under both well-watered and moderate drought-stressed conditions. During drought, AOX knockdown lines displayed a lower rate of respiration in the light than the wild type, as confirmed by two independent methods. Furthermore, CO₂ and light response curves indicated a nonstomatal limitation of photosynthesis in the knockdowns during drought, relative to the wild type. Also relative to the wild type, the knockdowns under drought maintained PSI and PSII in a more reduced redox state, showed greater regulated nonphotochemical energy quenching by PSII, and displayed a higher relative rate of cyclic electron transport around PSI. The origin of these differences may lie in the chloroplast ATP synthase amount, which declined dramatically in the knockdowns in response to drought. None of these effects were seen in plants overexpressing AOX. The results show that AOX is necessary to maintain mitochondrial respiration during moderate drought. In its absence, respiration rate slows and the lack of this electron sink feeds back on the photosynthetic apparatus, resulting in a loss of chloroplast ATP synthase that then limits photosynthetic capacity.The plant mitochondrial electron transport chain (ETC) is bifurcated such that electrons in the ubiquinone pool partition between the cytochrome (cyt) pathway (consisting of Complex III, cyt c, and Complex IV) and alternative oxidase (AOX; Finnegan et al., 2004; Millar et al., 2011; Vanlerberghe, 2013). AOX directly couples ubiquinol oxidation with O₂ reduction to water. This reduces the energy yield of respiration because, unlike Complexes III and IV, AOX is not proton pumping. Hence, AOX is an electron sink, the capacity of which is little encumbered by rates of ATP turnover. In this way, AOX might be well suited to prevent cellular over-reduction. Supporting this, transgenic Nicotiana tabacum leaves with suppressed amounts of AOX have increased concentrations of mitochondrial-localized superoxide radical (O₂−) and nitric oxide, the products that can arise when an over-reduced ETC results in electron leakage to O₂ or nitrite (Cvetkovska and Vanlerberghe, 2012, 2013).In angiosperms, AOX is encoded by a small gene family (Considine et al., 2002). In Arabidopsis (Arabidopsis thaliana), mutation or knockdown of the stress-responsive AOX1a gene family member dramatically reduces AOX protein and the capacity of the AOX respiration pathway to consume O₂. Several studies have shown that this loss of AOX capacity in Arabidopsis aox1a plants affected processes such as growth, carbon and energy metabolism, and/or the cellular network of reactive oxygen species (ROS) scavengers (Fiorani et al., 2005; Umbach et al., 2005; Watanabe et al., 2008; Giraud et al., 2008; Skirycz et al., 2010). However, in studies in which respiration was measured, it was consistently reported that the lack of AOX capacity had no significant impact on the respiration rate in the dark (R_D; Umbach et al., 2005; Giraud et al., 2008; Strodtkötter et al., 2009; Florez-Sarasa et al., 2011; Yoshida et al., 2011b; Gandin et al., 2012). The exceptions are two reports that R_D was actually higher in aox1a than in the wild type under some conditions (Watanabe et al., 2008; Vishwakarma et al., 2014). To our knowledge, how the lack of AOX affects respiration rate in the light (R_L) is not reported in Arabidopsis or other species.Numerous studies have established the importance of mitochondrial metabolism in the light to optimize photosynthesis (Hoefnagel et al., 1998; Raghavendra and Padmasree, 2003). In recent years, the potential importance of specifically AOX respiration during photosynthesis has been examined using the Arabidopsis aox1a plants (Giraud et al., 2008; Strodtkötter et al., 2009; Zhang et al., 2010; Florez-Sarasa et al., 2011; Yoshida et al., 2011a, 2011b). In general, these studies reported small perturbations of photosynthesis in standard-grown aox1a plants, including slightly lower rates of CO₂ uptake or O₂ release (Gandin et al., 2012; Vishwakarma et al., 2014), slightly higher rates of cyclic electron transport (CET; Yoshida et al., 2011b), and slightly increased susceptibility to photoinhibition after a high light treatment (Florez-Sarasa et al., 2011). Generally, these studies concluded that aox1a plants exhibit a biochemical limitation of photosynthesis, in line with the hypothesis that AOX serves as a sink for excess photogenerated reducing power, with the reductant likely reaching the mitochondrion via the malate valve (Noguchi and Yoshida, 2008; Taniguchi and Miyake, 2012). Similar to these Arabidopsis studies, we recently reported that well-watered N. tabacum AOX knockdowns grown at moderate irradiance display a slight reduced rate of photosynthesis (approximately 10%–15%) when measured at high irradiance. However, we established that the lower photosynthetic rate was the result of a stomatal rather than biochemical limitation of photosynthesis, and provided evidence that this stomatal limitation resulted from disrupted nitric oxide homeostasis within the guard cells of AOX knockdown plants (Cvetkovska et al., 2014).Drought is a common abiotic stress that can substantially curtail photosynthesis because stomatal closure, meant to conserve water, also restricts CO₂ availability to the Calvin cycle. Besides this well established stomatal limitation of photosynthesis, there may also be water deficit-sensitive biochemical components that contribute to the reduction of photosynthesis during drought. However, the nature of this biochemical limitation and the degree to which it contributes to the curtailment of photosynthesis during drought remain areas of active debate (Flexas et al., 2004; Lawlor and Tezara, 2009; Pinheiro and Chaves, 2011). Additional factors, such as patchy stomatal closure (Sharkey and Seemann, 1989; Gunasekera and Berkowitz, 1992) or changes in the conductance to CO₂ of mesophyll cells (Perez-Martin et al., 2009), can further complicate analyses of photosynthesis during drought.Metabolism can experience energy imbalances, when there is a mismatch between rates of synthesis and rates of utilization of ATP and/or NADPH, and the importance of mechanisms to minimize such imbalances has been emphasized (Cruz et al., 2005; Kramer and Evans, 2011; Vanlerberghe, 2013). For example, such imbalances may occur in the chloroplast when the use of ATP and NADPH by the Calvin cycle does not keep pace with the harvesting of light energy (Hüner et al., 2012). This can result in excess excitation energy that can damage photosynthetic components, perhaps through the generation of ROS (Asada, 2006; Noctor et al., 2014). Such a scenario has been hypothesized to underlie the development of the biochemical limitations of photosynthesis reported during drought (Lawlor and Tezara, 2009).In this study, we find that N. tabacum AOX knockdowns show a compromised rate of mitochondrial respiration in the light during moderate drought. This corresponds with a strong nonstomatal limitation of photosynthesis in these plants relative to the wild type, and we describe a biochemical basis for this photosynthetic limitation. The results indicate that AOX is a necessary electron sink to support photosynthesis during drought, a condition when the major photosynthetic electron sink, the Calvin cycle, is becoming limited by CO₂ availability.     

Elevated Temperature and CO2 Stimulate Late-Season Photosynthesis But Impair Cold Hardening in Pine

Rising global temperature and CO₂ levels may sustain late-season net photosynthesis of evergreen conifers but could also impair the development of cold hardiness. Our study investigated how elevated temperature, and the combination of elevated temperature with elevated CO₂, affected photosynthetic rates, leaf carbohydrates, freezing tolerance, and proteins involved in photosynthesis and cold hardening in Eastern white pine (Pinus strobus). We designed an experiment where control seedlings were acclimated to long photoperiod (day/night 14/10 h), warm temperature (22°C/15°C), and either ambient (400 μL L⁻¹) or elevated (800 μmol mol⁻¹) CO₂, and then shifted seedlings to growth conditions with short photoperiod (8/16 h) and low temperature/ambient CO₂ (LTAC), elevated temperature/ambient CO₂ (ETAC), or elevated temperature/elevated CO₂ (ETEC). Exposure to LTAC induced down-regulation of photosynthesis, development of sustained nonphotochemical quenching, accumulation of soluble carbohydrates, expression of a 16-kD dehydrin absent under long photoperiod, and increased freezing tolerance. In ETAC seedlings, photosynthesis was not down-regulated, while accumulation of soluble carbohydrates, dehydrin expression, and freezing tolerance were impaired. ETEC seedlings revealed increased photosynthesis and improved water use efficiency but impaired dehydrin expression and freezing tolerance similar to ETAC seedlings. Sixteen-kilodalton dehydrin expression strongly correlated with increases in freezing tolerance, suggesting its involvement in the development of cold hardiness in P. strobus. Our findings suggest that exposure to elevated temperature and CO₂ during autumn can delay down-regulation of photosynthesis and stimulate late-season net photosynthesis in P. strobus seedlings. However, this comes at the cost of impaired freezing tolerance. Elevated temperature and CO₂ also impaired freezing tolerance. However, unless the frequency and timing of extreme low-temperature events changes, this is unlikely to increase risk of freezing damage in P. strobus seedlings.Land surface temperature is increasing, particularly in the northern hemisphere (IPCC, 2014), which is dominated by boreal and temperate forests. At higher latitudes, trees rely on temperature and photoperiod cues to detect changing seasons and to trigger cessation of growth and cold hardening during the autumn (Ensminger et al., 2015). For boreal and temperate evergreen conifers, cold hardening involves changes in carbohydrate metabolism, down-regulation of photosynthesis, accumulation of cryoprotective metabolites, and development of freezing tolerance (Crosatti et al., 2013; Ensminger et al., 2015). These processes minimize freezing damage and enable conifers to endure winter stresses. However, rising temperatures result in asynchronous phasing of temperature and photoperiod characterized by delayed arrival of first frosts (McMahon et al., 2010), which may impact the onset and development of cold hardening during autumn.Short photoperiod induces the cessation of growth in many tree species (Downs and Borthwick, 1956; Heide, 1974; Repo et al., 2000; Böhlenius et al., 2006). As a consequence, carbon demand in sink tissue decreases toward the end of the growing season, and the bulk of photoassimilate is translocated from source tissues to storage tissues (Hansen and Beck, 1994; Oleksyn et al., 2000). In addition, cryoprotective soluble sugars, including sucrose, raffinose, and pinitol, accumulate in leaf tissues to enhance freezing tolerance (Strimbeck et al., 2008; Angelcheva et al., 2014). Thus, by winter, leaf nonstructural carbohydrates are mainly comprised of mono- and oligosaccharides, and only minimal levels of starch remain (Hansen and Beck, 1994; Strimbeck et al., 2008). The concurrent decrease of photoassimilate and demand for metabolites that occur during the cessation of growth also impacts the citric acid cycle that mediates between photosynthesis, respiration, and protein synthesis. The citric acid cycle generates NADH to fuel ATP synthesis via mitochondrial electron transport, as well as amino acid precursors (Shi et al., 2015). In C3 plants, the enzyme phosphoenolpyruvate carboxylase (PEPC) converts phosphoenolpyruvate to oxaloacetic acid in order to supplement the flow of metabolites to the citric acid cycle and thus controls the regulation of respiration and photosynthate partitioning (O’Leary et al., 2011).Cessation of growth, low temperature, and presumably short photoperiod decrease the metabolic sink for photoassimilates, resulting in harmful excess light energy (Öquist and Huner, 2003; Ensminger et al., 2006) and increased generation of reactive oxygen species (Adams et al., 2004). During autumn and the development of cold hardiness, conifers reconfigure the photosynthetic apparatus in order to avoid formation of excess light and reactive oxygen species. This involves a decrease in chlorophylls and PSII reaction center core protein D1 (Ottander et al., 1995; Ensminger et al., 2004; Verhoeven et al., 2009), as well as aggregation of light-harvesting complex proteins (Ottander et al., 1995; Busch et al., 2007). Additionally, photoprotective carotenoid pigments accumulate in leaves, especially the xanthophylls, zeaxanthin, and lutein that contribute to nonphotochemical quenching (NPQ) via thermal dissipation of excess light energy (Busch et al., 2007; Verhoeven et al., 2009; Demmig-Adams et al., 2012). Prolonged exposure to low temperature induces sustained nonphotochemical quenching (NPQ_S), where zeaxanthin constitutively dissipates excess light energy (Ensminger et al., 2004; Demmig-Adams et al., 2012; Fréchette et al., 2015).In conifers, freezing tolerance is initiated during early autumn in response to decreasing photoperiod (Rostad et al., 2006; Chang et al., 2015) and continues to develop through late autumn in response to the combination of short photoperiod and low temperature (Strimbeck and Schaberg, 2009; Chang et al., 2015). In addition to changes in carbohydrate content, freezing tolerance also involves the expression of specific dehydrins (Close, 1997; Kjellsen et al., 2013). Members of the dehydrin protein family are involved in responses to osmotic, salt, and freezing stress (Close, 1996). Dehydrins have been associated with improved freezing tolerance in many species including spinach (Kaye et al., 1998), strawberry (Houde et al., 2004), cucumber (Yin et al., 2006), peach (Wisniewski et al., 1999), birch (Puhakainen et al., 2004), and spruce (Kjellsen et al., 2013). In angiosperms, a characteristic Lys-rich dehydrin motif known as the K-segment interacts with lipids to facilitate membrane binding (Koag et al., 2003; Eriksson et al., 2011). Several in vitro studies have demonstrated dehydrin functions including prevention of aggregation and unfolding of enzymes (using Vitis riparia; Hughes and Graether, 2011), radical scavenging (using Citrus unshiu; Hara et al., 2004), and suppression of ice crystal formation (using Prunus persica; Wisniewski et al., 1999). To date, dehydrin functions have not been demonstrated in planta.Rising temperatures since the mid-twentieth century have delayed the onset of autumn dormancy and increased length of the growing season in forests across the northern hemisphere (Boisvenue and Running, 2006; Piao et al., 2007; McMahon et al., 2010). Studies have shown that elevated temperatures ranging from +4°C to +20°C above ambient can delay down-regulation of photosynthesis in several evergreen conifers. Consistent findings were apparent among climate-controlled chamber studies exposing Pinus strobus seedlings to a sudden shift in temperature and/or photoperiod (Fréchette et al., 2016), as well as chamber studies exposing Picea abies seedlings to simulated autumn conditions using a gradient of decreasing temperature and photoperiod (Stinziano et al., 2015). Similar findings were also demonstrated in open-top chamber experiments exposing mature Pinus sylvestris to a gradient of decreasing temperature and natural photoperiod (Wang, 1996). Elevated temperature (+4°C above ambient) also impaired cold hardening in Pseudotsuga menziesii seedlings (Guak et al., 1998) and mature P. sylvestris (Repo et al., 1996) exposed to a decreasing gradient of temperature and natural photoperiod using open-top chambers. In contrast, a recent study showed that smaller temperature increments (+1.5°C to +3°C) applied using infrared heaters did not delay down-regulation of photosynthesis or impair freezing tolerance in field-grown P. strobus seedlings that were acclimated to larger diurnal and seasonal temperature variations (Chang et al., 2015). For many tree species, photoperiod determines cessation of growth (Tanino et al., 2010; Petterle et al., 2013), length of the growing season (Bauerle et al., 2012), and development of cold hardiness (Welling et al., 1997; Li et al., 2003; Rostad et al., 2006). However, the effects of climate warming on tree phenology are complex and can be unpredictable due to species- and provenance-specific differences in sensitivity to photoperiod and temperature cues (Körner and Basler, 2010; Basler and Körner, 2012; Basler and Körner, 2014).The effect of elevated CO₂ further increases uncertainties in the response of trees to warmer climate. Similar to warmer temperature, elevated CO₂ may also delay the down-regulation of photosynthesis in evergreens and extend the length of the growing season, as demonstrated in mature P. sylvestris (Wang, 1996). Elevated CO₂ increases carbon assimilation (Curtis and Wang, 1998; Ainsworth and Long, 2005) and biomass production (Ainsworth and Long, 2005) during the growing season. The effects could continue during the autumn if dormancy or growth cessation is delayed, which suggests that elevated CO₂ may increase annual carbon uptake. However, long-term exposure to elevated CO₂ can also down-regulate photosynthesis during the growing season (Ainsworth and Long, 2005). Prior studies that have attempted to determine the impact of a combination of elevated CO₂ and/or temperature on cold hardening in evergreens have largely focused on freezing tolerance, with contrasting results. Open-top chamber experiments showed that a combination of elevated temperature and CO₂ both delayed and impaired freezing tolerance of P. menziesii seedlings (Guak et al., 1998) and evergreen broadleaf Eucalyptus pauciflora seedlings (Loveys et al., 2006) but did not affect freezing tolerance of mature P. sylvestris (Repo et al., 1996). A recent field experiment examining mature trees revealed that Larix decidua, but not Pinus mugo, exhibited enhanced freezing damage following six years of exposure to combined soil warming and elevated CO₂ (Rixen et al., 2012). In contrast, a climate-controlled study showed that exposure to elevated CO₂ advanced the date of bud set and improved freezing tolerance in Picea mariana seedlings (Bigras and Bertrand, 2006). In a second study on similar seedlings conducted by the same authors, exposure of trees to elevated CO₂ also enhanced freezing tolerance but impaired the accumulation of sucrose and raffinose (Bertrand and Bigras, 2006). These previous experiments used experimental conditions where temperature and photoperiod gradually decreased. While this approach aims to mimic natural conditions, it is difficult to distinguish specific responses to either photoperiod or temperature. Because of the contrasting findings from previous studies, we designed an experiment aiming to separate the effects of photoperiod, temperature, and CO₂ on a wide range of parameters that are involved in cold hardening in conifers.Our study aimed to determine (1) how induction and development of the cold hardening process is affected by a shift from long to short photoperiod under warm conditions and (2) how the combination of warm air temperature and elevated CO₂ affects photoperiod-induced cold hardening processes in Eastern white pine (P. strobus). To assess the development of cold hardening, we measured photosynthetic rates, changes in leaf carbohydrates, freezing tolerance, and proteins involved in photosynthesis and cold hardening over 36 d. Assuming that both low temperature and short photoperiod cues are required to induce cold hardening in conifers, we hypothesized that warm temperature and the combination of warm temperature and elevated CO₂ would prevent seedlings growing under autumn photoperiod from down-regulating photosynthesis. We further hypothesized that warm temperature and the combination of warm temperature and elevated CO₂ would impair the development of freezing tolerance, due to a lack of adequate phasing of the low temperature and short photoperiod signals.     

Engineering Triterpene and Methylated Triterpene Production in Plants Provides Biochemical and Physiological Insights into Terpene Metabolism

Linear, branch-chained triterpenes, including squalene (C30), botryococcene (C30), and their methylated derivatives (C31–C37), generated by the green alga Botryococcus braunii race B have received significant attention because of their utility as chemical and biofuel feedstocks. However, the slow growth habit of B. braunii makes it impractical as a production system. In this study, we evaluated the potential of generating high levels of botryococcene in tobacco (Nicotiana tabacum) plants by diverting carbon flux from the cytosolic mevalonate pathway or the plastidic methylerythritol phosphate pathway by the targeted overexpression of an avian farnesyl diphosphate synthase along with two versions of botryococcene synthases. Up to 544 µg g⁻¹ fresh weight of botryococcene was achieved when this metabolism was directed to the chloroplasts, which is approximately 90 times greater than that accumulating in plants engineered for cytosolic production. To test if methylated triterpenes could be produced in tobacco, we also engineered triterpene methyltransferases (TMTs) from B. braunii into wild-type plants and transgenic lines selected for high-level triterpene accumulation. Up to 91% of the total triterpene contents could be converted to methylated forms (C31 and C32) by cotargeting the TMTs and triterpene biosynthesis to the chloroplasts, whereas only 4% to 14% of total triterpenes were methylated when this metabolism was directed to the cytoplasm. When the TMTs were overexpressed in the cytoplasm of wild-type plants, up to 72% of the total squalene was methylated, and total triterpene (C30+C31+C32) content was elevated 7-fold. Altogether, these results point to innate mechanisms controlling metabolite fluxes, including a homeostatic role for squalene.Terpenes and terpenoids represent a distinct class of natural products (Buckingham, 2003) that are derived from two universal five-carbon precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In eukaryotic fungi and animals, IPP and DMAPP are synthesized via the mevalonate (MVA) pathway, whereas in prokaryotes, they are synthesized via the methylerythritol phosphate (MEP) pathway. In higher plants, the pathways are present in separate compartments and are believed to operate independently. The MVA pathway in the cytoplasm is predominantly responsible for sesquiterpene (C15), triterpene (C30), and polyprenol (greater than C45) biosynthesis and associated with the endoplasmic reticulum (ER) system. The MEP pathway resides in plastids and is dedicated to monoterpenes (C10), diterpenes (C20), carotenoids (C40), and long-chain phytol biosynthesis. All these compounds are usually produced by plants for a variety of physiological (i.e. hormones, aliphatic membrane anchors, and maintaining membrane structure) and ecological (i.e. defense compounds and insect/animal attractants) roles (Kempinski et al., 2015). Terpenes are also important for various industrial applications, ranging from flavors and fragrances (Schwab et al., 2008) to medicines (Dewick, 2009; Niehaus et al., 2011; Shelar, 2011).The utility of terpenes as chemical and biofuel feedstocks has also received considerable attention recently. Isoprenoid-derived biofuels include farnesane (Renninger and McPhee, 2008; Rude and Schirmer, 2009), bisabolene (Peralta-Yahya et al., 2011), pinene dimers (Harvey et al., 2010), isopentenal (Withers et al., 2007), and botryococcene (Moldowan and Seifert, 1980; Hillen et al., 1982; Glikson et al., 1989; Mastalerz and Hower, 1996). The richness of branches within these hydrocarbon scaffolds correlate with their high-energy content, which enables them to serve as suitable alternatives to crude petroleum (Peralta-Yahya and Keasling, 2010). Indeed, some of them are already major contributors to current-day petroleum-based fuels. One of the best examples of this is the triterpene oil accumulating in the green alga Botryococcus braunii race B, which is considered a major progenitor to oil and coal shale deposits (Moldowan and Seifert, 1980). This alga has been well studied, and the major constituents of its prodigious hydrocarbon oil are a group of triterpenes including squalene (C30), organism-specific botryococcene (C30), methylated squalene (C31–C34), and methylated botryococcene (C31–C37; Metzger et al., 1988; Huang and Poulter, 1989; Okada et al., 1995), which can be readily converted into all classes of combustible fuels under hydrocracking conditions (Hillen et al., 1982).The unique biosynthetic mechanism for the triterpenes in B. braunii was recently described by Niehaus et al. (2011), and a series of novel squalene synthase-like genes were identified (Fig. 1). In short, squalene synthase-like enzyme, SSL-1, performs a head-to-head condensation of two farnesyl diphosphate (FPP) molecules into presqualene diphosphate, followed by a reductive rearrangement to yield squalene (C30) by the enzyme SSL-2, or is converted by SSL-3 to form botryococcene through a different reductive rearrangement (Niehaus et al., 2011). Methylated derivatives are the dominant triterpene species generated by B. braunii race B (Metzger, 1985; Metzger et al., 1988), and these derivatives are known to yield higher quality fuels due to their high energy content and the hydrocracking products derived by virtue of having more hydrocarbon branches. Triterpene methyltransferases (TMTs) that can methylate squalene and botryococcene have been successfully characterized by Niehaus et al. (2012). TRITERPENE METHYLTRANSFERASE1 (TMT-1) and TMT-2 prefer squalene C30 as their substrate for the production of monomethylated (C31) or dimethylated (C32) squalene, while TMT-3 prefers botryococcene as its substrate for the biosynthesis of monomethylated (C31) or dimethylated (C32) botryococcene (Fig. 1). These TMTs are believed to be insoluble enzymes; they exhibit large hydrophobic areas, and their activities were only observed in vitro using yeast microsomal preparations (no activity was observed when expressed in bacteria; Niehaus et al., 2012).Open in a separate windowFigure 1.Depiction of the catalytic roles of novel SSL and TMT enzymes in B. braunii race B and their putative contributions to the triterpene constituents (Niehaus et al., 2011; Niehaus et al., 2012). SSL-1 catalyzes the condensation of two farnesyl diphosphate (FPP) molecules to presqualene diphosphate (PSPP), which is converted to either squalene or botryococcene by SSL-2 or SSL-3, respectively. Squalene can also be synthesized directly from the condensation of two FPP molecules catalyzed by squalene synthase (SQS). TMT-1 and TMT-2 transfer the methyl donor group from S-adenosylmethionine (SAM) to squalene to form monomethylated and dimethylated squalene, whereas TMT-3 acts on botryococcene to form monomethylated and dimethylated botryococcene (Niehaus et al., 2012).Like the majority of identified methyltransferases, these TMTs utilize the methyl donor S-adenosyl methionine (SAM), which is ubiquitous in prokaryotes and eukaryotes (Scheer et al., 2011; Liscombe et al., 2012). In plants, SAM is one of the most abundant cofactors (Fontecave et al., 2004; Sauter et al., 2013) and is synthesized exclusively in the cytosol (Wallsgrove et al., 1983; Ravanel et al., 1998, 2004; Bouvier et al., 2006). While it is used predominantly as a methyl donor in the methylation reaction (Ravanel et al., 2004), it also serves as the primary precursor for the biosynthesis of ethylene (Wang et al., 2002b), polyamines (Kusano et al., 2008), and nicotianamine (Takahashi et al., 2003), which play a variety of important roles for plant growth and development (Huang et al., 2012; Sauter et al., 2013). The SAM present in organelles, like the chloroplast, appears to be imported from the cytosol by specific SAM/S-adenosylhomocysteine exchange transporters that reside on the envelope membranes of plastids (Ravanel et al., 2004; Bouvier et al., 2006). The imported SAM is involved in the biogenesis of Asp-derived amino acids (Curien et al., 1998; Jander and Joshi, 2009; Sauter et al., 2013) and serves as the methyl donor for the methylation of macromolecules, such as plastid DNA (Nishiyama et al., 2002; Ahlert et al., 2009) and proteins (Houtz et al., 1989; Niemi et al., 1990; Ying et al., 1999; Trievel et al., 2003; Alban et al., 2014), and small molecule metabolites, such as prenylipids (e.g. plastoquinone, tocopherol, chlorophylls, and phylloquinone; Bouvier et al., 2005, 2006; DellaPenna, 2005).Although plants and microbes are the natural sources for useful terpenes, most of them are produced in very small amounts and often as complex mixtures. In contrast, B. braunii produces large quantities of triterpenes, but its slow growth makes it undesirable as a viable production platform (Niehaus et al., 2011). Nevertheless, metabolic engineering and synthetic biology offer many strategies to manipulate terpene metabolism in various biological systems to achieve high-value terpene production with high yield and high fidelity for particular practical applications (Nielsen and Keasling, 2011). Many successes have been achieved in engineering valuable terpenes in heterotrophic microbes, such as Escherichia coli (Nishiyama et al., 2002; Martin et al., 2003; Ajikumar et al., 2010) and Saccharomyces cerevisiae (Ro et al., 2006; Takahashi et al., 2007; Westfall et al., 2012; Zhuang and Chappell, 2015). The strategies developed in these efforts usually take advantage of specific microbe strains whose innate biosynthetic machinery is genetically modified to accumulate certain prenyldiphosphate precursors (e.g. IPP or FPP), which can be utilized by other introduced terpene synthase(s) for the production of the desired terpene(s). For example, greater than 900 mg L⁻¹ bisabolene was produced when bisabolene synthase genes from plants were introduced into FPP-overproducing E. coli or S. cerevisiae strains (Peralta-Yahya et al., 2011). High levels of farnesane production for diesel fuels were also achieved by reductive hydrogenation of its precursor farnesene, which was generated from a genetically engineered yeast (e.g. Saccharomyces cerevisiae) strain using plant farnesene synthases (Renninger and McPhee, 2008; Ubersax and Platt, 2010). However, terpene production using microbial platforms is still dependent on exogenous feedstocks (i.e. sugars) and elaborate production facilities, both of which add significantly to their production costs.Compared with microbial systems, engineering terpene production in plant systems seems like an attractive target as well. This is because plants can take advantage of photosynthesis by using atmospheric CO₂ as their carbon resource instead of relying on exogenous carbon feedstocks. Moreover, crop plants such as tobacco (Nicotiana tabacum) can generate a large amount of green tissues efficiently when grown for biomass production (Schillberg et al., 2003; Andrianov et al., 2010), making them a robust, sustainable, and scalable platform for large-scale terpene production. Nonetheless, compared with microbial platforms, there are only a few examples of elevating terpene production in bioengineered plants. This is due partly to higher plants being complex multicellular organisms, in which terpene metabolism generally utilizes more complex innate machinery that can be compartmentalized intracellularly and to cell/tissue specificities (Lange and Ahkami, 2013; Kempinski et al., 2015). Significant efforts have been made to overcome these obstacles to improve the production of valuable terpenes in plants, including monoterpenes (Lücker et al., 2004; Ohara et al., 2010; Lange et al., 2011), sesquiterpenes (Aharoni et al., 2003; Kappers et al., 2005; Wu et al., 2006; Davidovich-Rikanati et al., 2008), diterpenes (Besumbes et al., 2004; Anterola et al., 2009), and triterpenes (Inagaki et al., 2011; Wu et al., 2012). Among these, engineering terpene metabolism into a subcellular organelle, where the engineered enzymes/pathways can utilize unlimited/unregulated precursors as substrates, appears most successful. For example, Wu et al. (2006, 2012) expressed an avian farnesyl diphosphate synthase (FPS) with foreign sesquiterpene/triterpene synthases targeted to the plastid to divert the IPP/DMAPP pool from the plastidic MEP pathway to synthesize high levels of the novel sesquiterpenes patchoulol and amorpha-4,11-diene up to 30 µg g⁻¹ fresh weight and the triterpene squalene up to 1,000 µg g⁻¹ fresh weight. This strategy appears to be particularly robust because it avoids possible endogenous regulation of sesquiterpene and triterpene biosynthesis, which occurs normally in the cytoplasm, and relies upon more plastic precursor pools of IPP/DMAPP inherent in the plastid, which are primarily derived from the local CO₂ fixation (Wright et al., 2014).The goal of this study was to evaluate the prospects for engineering advanced features of triterpene metabolism from B. braunii into tobacco and, thus, to probe the innate intricacies of isoprenoid metabolism in plants. In order to achieve this, we first introduced the key steps of botryococcene biosynthesis into specific subcellular compartments of tobacco cells under the direction of constitutive or trichome-specific promoters. The transgenic lines expressing the enzymes in the chloroplast were found to accumulate the highest levels of botryococcene. Triterpene methyltransferases were next introduced into the same intracellular compartments of selected high-triterpene-accumulating lines. A high yield of methylated triterpenes was also achieved in transgenic lines when the TMTs were targeted to the chloroplast. Through careful comparison of the levels of triterpenes and the methylated triterpene products in the various transgenic lines, we have also gained a deeper insight into the subcellular distribution of the triterpene products in these transgenic lines as well as a better understanding of methylation metabolism for specialized metabolites in particular compartments. These findings all contribute to our understanding of the regulatory elements that control carbon flux through the innate terpene biosynthetic pathways operating in plants.