The Operation of Two Decarboxylases,Transamination, and Partitioning of C4 Metabolic Processes between Mesophyll and Bundle Sheath Cells Allows Light Capture To Be Balanced for the Maize C4 Pathway

The C₄ photosynthesis carbon-concentrating mechanism in maize (Zea mays) has two CO₂ delivery pathways to the bundle sheath (BS; via malate or aspartate), and rates of phosphoglyceric acid reduction, starch synthesis, and phosphoenolpyruvate regeneration also vary between BS and mesophyll (M) cells. The theoretical partitioning of ATP supply between M and BS cells was derived for these metabolic activities from simulated profiles of light penetration across a leaf, with a potential 3-fold difference in the fraction of ATP produced in the BS relative to M (from 0.29 to 0.96). A steady-state metabolic model was tested using varying light quality to differentially stimulate M or BS photosystems. CO₂ uptake, ATP production rate (J_ATP; derived with a low oxygen/chlorophyll fluorescence method), and carbon isotope discrimination were measured on plants under a low light intensity, which is considered to affect C₄ operating efficiency. The light quality treatments did not change the empirical ATP cost of gross CO₂ assimilation (J_ATP/GA). Using the metabolic model, measured J_ATP/GA was compared with the predicted ATP demand as metabolic functions were varied between M and BS. Transamination and the two decarboxylase systems (NADP-malic enzyme and phosphoenolpyruvate carboxykinase) were critical for matching ATP and reduced NADP demand in BS and M when light capture was varied under contrasting light qualities.Interest in the C₄ pathway has been increased by the potential for enhancing crop productivity and maintaining yield stability in the face of global warming and population pressure (Friso et al., 2010; Zhu et al., 2010; Covshoff and Hibberd, 2012). Maize (Zea mays), a C₄ plant of the NADP-malic enzyme (ME) subtype, is a leading grain production cereal (www.fao.org). C₄ photosynthesis is a shared activity between mesophyll (M; abbreviations are listed in BS) cells, coupled to allow the operation of a biochemical carbon-concentrating mechanism (CCM). The CCM effectively minimizes photorespiration by increasing the CO₂ concentration in the bundle sheath (C_BS), where Rubisco is exclusively expressed. Since BS and M are connected by plasmodesmata, some CO₂ retrodiffuses. The refixation of that escaping CO₂ by the CCM increases the activity of the CCM and the total ATP demand (ATP_BS + ATP_M) for gross CO₂ assimilation (GA; [ATP_BS + ATP_M]/GA), from a theoretical minimum of five ATPs (Furbank et al., 1990). Leakiness (Φ), the amount of CO₂ retrodiffusing relative to phosphoenolpyruvate (PEP) carboxylation rate, is therefore a proxy for the coordination between the CCM and assimilatory activity (Henderson et al., 1992; Tazoe et al., 2008; Kromdijk et al., 2010; Ubierna et al., 2011; Bellasio and Griffiths, 2013).

Table I.

Variables and acronyms described in the text

Coordination of Leaf Photosynthesis,Transpiration, and Structural Traits in Rice and Wild Relatives (Genus Oryza)

The genus Oryza, which includes rice (Oryza sativa and Oryza glaberrima) and wild relatives, is a useful genus to study leaf properties in order to identify structural features that control CO₂ access to chloroplasts, photosynthesis, water use efficiency, and drought tolerance. Traits, 26 structural and 17 functional, associated with photosynthesis and transpiration were quantified on 24 accessions (representatives of 17 species and eight genomes). Hypotheses of associations within, and between, structure, photosynthesis, and transpiration were tested. Two main clusters of positively interrelated leaf traits were identified: in the first cluster were structural features, leaf thickness (Thick_leaf), mesophyll (M) cell surface area exposed to intercellular air space per unit of leaf surface area (S_mes), and M cell size; a second group included functional traits, net photosynthetic rate, transpiration rate, M conductance to CO₂ diffusion (g_m), stomatal conductance to gas diffusion (g_s), and the g_m/g_s ratio. While net photosynthetic rate was positively correlated with g_m, neither was significantly linked with any individual structural traits. The results suggest that changes in g_m depend on covariations of multiple leaf (S_mes) and M cell (including cell wall thickness) structural traits. There was an inverse relationship between Thick_leaf and transpiration rate and a significant positive association between Thick_leaf and leaf transpiration efficiency. Interestingly, high g_m together with high g_m/g_s and a low S_mes/g_m ratio (M resistance to CO₂ diffusion per unit of cell surface area exposed to intercellular air space) appear to be ideal for supporting leaf photosynthesis while preserving water; in addition, thick M cell walls may be beneficial for plant drought tolerance.Leaves have evolved in different environments into a multitude of sizes and shapes, showing great variation in morphology and anatomy (Evans et al., 2004). However, all leaf typologies share common functions associated with chloroplasts, namely to intercept sunlight, take up CO₂ and inorganic nitrogen, and perform photosynthesis as a primary process for growth and reproduction.Investigating relationships between leaf anatomy and photosynthetic features (CO₂ fixation, which involves physical and biochemical processes and loss of water by transpiration) could lead to the identification of structural features for enhancing crop productivity and improve our understanding of plant evolution and adaptation (Evans et al., 2004).Stomata, through which CO₂ and water vapor diffuse into and out of the leaf, are involved in the regulation and control of photosynthetic and transpiration responses (Jarvis and Morison, 1981; Farquhar and Sharkey, 1982). Besides stomata distribution patterns between the abaxial and adaxial lamina surfaces (Foster and Smith, 1986), stomatal density and size are leaf anatomical traits contributing to build the leaf stomatal conductance to gas diffusion (g_s). This is calculated as the reciprocal of the stomatal resistances to gas diffusion; stomatal control results in a lower concentration of CO₂ in the leaf mesophyll (M) intercellular air space (C_i) than in the atmosphere (C_a; Nobel, 2009).Leaf M architecture greatly contributes to the pattern of light attenuation profiles within the lamina (Terashima and Saeki, 1983; Woolley, 1983; Vogelmann et al., 1989; Evans, 1999; Terashima et al., 2011) and affects CO₂ diffusion from the intercellular air space (IAS) to the chloroplast stroma. Therefore, it influences photosynthetic activity (Flexas et al., 2007, 2008) and can have effects on leaf hydrology and transpiration (Sack et al., 2003; Brodribb et al., 2010; Ocheltree et al., 2012). In addition, M architecture sets boundaries for leaf photosynthetic responses to changing environmental conditions (Nobel et al., 1975).Fortunately, several methodologies are currently available (Flexas et al., 2008; Pons et al., 2009) to determine M conductance to CO₂ diffusion (g_m), expressed per unit of leaf surface area. It is calculated as the reciprocal of the cumulated partial resistances exerted by leaf structural traits and biochemical processes from the substomatal cavities to photosynthetic sites (Evans et al., 2009; Nobel, 2009). The resistance to CO₂ diffusion in the liquid phase is 4 orders of magnitude higher than in the gaseous phase (Nobel, 2009); therefore, the changes in CO₂ concentration in the leaf gas phase are small in comparison with the changes in the liquid phase (Niinemets, 1999; Aalto and Juurola, 2002; Nobel, 2009). In the liquid phase, the resistance to CO₂ transfer is built from contributions by the cell walls, the plasmalemma, cytoplasm, chloroplast membranes, and stroma (Tholen and Zhu, 2011; Tholen et al., 2012); in addition, it involves factors associated with the carboxylation reaction (Kiirats et al., 2002; Evans et al., 2009). Thus, the concentration of CO₂ in the chloroplasts (C_c) is lower than C_i and can limit photosynthesis.At steady state, the relationships between the leaf net photosynthetic rate (A), the concentrations of CO₂, and the stomatal conductance to CO₂ diffusion (g_{s_CO2}) and g_m are modeled based on Fick’s first law of diffusion (Nobel, 2009) as:(1)where C_a, C_i, and C_c are as defined above (Flexas et al., 2008).The magnitude of g_m has been found to correlate with certain leaf structural traits in some species, in particular with the M cell surface area exposed to IAS per (one side) unit of leaf surface area (S_mes) and its extent covered by chloroplasts (S_chl; Evans and Loreto, 2000; Slaton and Smith, 2002; Tholen et al., 2012). From a physical modeling perspective, increasing S_mes provides more pathways acting in parallel for CO₂ diffusion (to and from the chloroplasts) per unit of leaf surface area; thus, it tends to reduce the resistance to CO₂ movement into the M cells and to increase g_m (Evans et al., 2009; Nobel, 2009). A number of leaf structural traits affect S_mes, including leaf thickness, cell density, cell volume and shape, and the fraction of the M cell walls in contact with the IAS (Terashima et al., 2001, 2011), and the degree they are linked to S_mes can vary between species (Slaton and Smith, 2002; Terashima et al., 2006). In particular, the presence of lobes on M cells, which are prominent in some Oryza species, may contribute to g_m through increasing S_mes (Sage and Sage, 2009; Terashima et al., 2011; Tosens et al., 2012). The M cell wall can provide resistance in series for M CO₂ diffusion (Nobel, 2009); thicker cell walls may increase resistance to CO₂ movement into the M cells and decrease g_m (Terashima et al., 2006, 2011; Evans et al., 2009).Other leaf traits, such as M porosity (the fraction of M volume occupied by air spaces [Vol_IAS]), has been shown to have a positive correlation with g_m in some species (Peña-Rojas et al., 2005), but the association may be mediated by light availability (Slaton and Smith, 2002). Leaf thickness (Thick_leaf) tends to be negatively linked to g_m, and it may set an upper limit for the maximum g_m, according to Terashima et al. (2006), Flexas et al. (2008), and Niinemets et al. (2009).With respect to leaf structural traits and water relations, Thick_leaf may increase the apoplast path length (resistances in series; Nobel, 2009) in the extra-xylem M (Sack and Holbrook, 2006; Brodribb et al., 2007) for water to reach the evaporation sites, which could decrease the conductance of water through the M and lower the transpiration rate. Interestingly, while thicker M cell walls may reduce g_m, they can enable the development of higher water potential gradients between the soil and leaves, which can be decisive for plant survival and longevity under drought conditions (Steppe et al., 2011).The purpose of this study was to provide insight into how the diversity of leaf structure relates to photosynthesis and transpiration among representative cultivated species and wild relatives in the genus Oryza. This includes, in particular, identifying leaf structural features associated with the diffusion of CO₂ from the atmosphere to the chloroplasts, photosynthesis, transpiration efficiency (A/E), and drought tolerance. The genus consists of 10 genomic groups and is composed of approximately 24 species (the number depending on taxonomic preferences; Kellogg, 2009; Brar and Singh, 2011), including the cultivated species Oryza sativa and Oryza glaberrima. Oryza species are distributed around the world, and they exhibit a wide range of phenotypes, with annual versus perennial life cycles and sun- versus shade-adapted species (Vaughan, 1994; Vaughan et al., 2008; Brar and Singh, 2011; Jagadish et al., 2011). This diversity in the genus is an important resource, which is being studied to improve rice yield, especially under unfavorable environmental conditions. In particular, O. glaberrima, Oryza australiensis, and Oryza meridionalis are of interest as drought-tolerant species (Henry et al., 2010; Ndjiondjop et al., 2010; Scafaro et al., 2011, 2012), while Oryza coarctata is salt tolerant (Sengupta and Majumder, 2010). In this study, a total of 43 leaf functional and structural parameters were collected on 24 accessions corresponding to 17 species within eight genomes (Brar and Singh (2011). Life cycle is as follows: A = annual; B = biennial; P = poliennial. Habitat is as follows: S = shade; S-Sh = sun-shade.

Focus on Metabolism: Posttranslational Protein Modifications in Plant Metabolism

Posttranslational modifications (PTMs) of proteins greatly expand proteome diversity, increase functionality, and allow for rapid responses, all at relatively low costs for the cell. PTMs play key roles in plants through their impact on signaling, gene expression, protein stability and interactions, and enzyme kinetics. Following a brief discussion of the experimental and bioinformatics challenges of PTM identification, localization, and quantification (occupancy), a concise overview is provided of the major PTMs and their (potential) functional consequences in plants, with emphasis on plant metabolism. Classic examples that illustrate the regulation of plant metabolic enzymes and pathways by PTMs and their cross talk are summarized. Recent large-scale proteomics studies mapped many PTMs to a wide range of metabolic functions. Unraveling of the PTM code, i.e. a predictive understanding of the (combinatorial) consequences of PTMs, is needed to convert this growing wealth of data into an understanding of plant metabolic regulation.The primary amino acid sequence of proteins is defined by the translated mRNA, often followed by N- or C-terminal cleavages for preprocessing, maturation, and/or activation. Proteins can undergo further reversible or irreversible posttranslational modifications (PTMs) of specific amino acid residues. Proteins are directly responsible for the production of plant metabolites because they act as enzymes or as regulators of enzymes. Ultimately, most proteins in a plant cell can affect plant metabolism (e.g. through effects on plant gene expression, cell fate and development, structural support, transport, etc.). Many metabolic enzymes and their regulators undergo a variety of PTMs, possibly resulting in changes in oligomeric state, stabilization/degradation, and (de)activation (Huber and Hardin, 2004), and PTMs can facilitate the optimization of metabolic flux. However, the direct in vivo consequence of a PTM on a metabolic enzyme or pathway is frequently not very clear, in part because it requires measurements of input and output of the reactions, including flux through the enzyme or pathway. This Update will start out with a short overview on the major PTMs observed for each amino acid residue (PTMs, including determination of the localization within proteins (i.e. the specific residues) and occupancy. Challenges in dealing with multiple PTMs per protein and cross talk between PTMs will be briefly outlined. We then describe the major physiological PTMs observed in plants as well as PTMs that are nonenzymatically induced during sample preparation (PTMs, in particular for enzymes in primary metabolism (Calvin cycle, glycolysis, and respiration) and the C4 shuttle accommodating photosynthesis in C4 plants (PTMs observed in plants

Variation in Adult Plant Phenotypes and Partitioning among Seed and Stem-Borne Roots across Brachypodium distachyon Accessions to Exploit in Breeding Cereals for Well-Watered and Drought Environments

Seedling roots enable plant establishment. Their small phenotypes are measured routinely. Adult root systems are relevant to yield and efficiency, but phenotyping is challenging. Root length exceeds the volume of most pots. Field studies measure partial adult root systems through coring or use seedling roots as adult surrogates. Here, we phenotyped 79 diverse lines of the small grass model Brachypodium distachyon to adults in 50-cm-long tubes of soil with irrigation; a subset of 16 lines was droughted. Variation was large (total biomass, ×8; total root length [TRL], ×10; and root mass ratio, ×6), repeatable, and attributable to genetic factors (heritabilities ranged from approximately 50% for root growth to 82% for partitioning phenotypes). Lines were dissected into seed-borne tissues (stem and primary seminal axile roots) and stem-borne tissues (tillers and coleoptile and leaf node axile roots) plus branch roots. All lines developed one seminal root that varied, with branch roots, from 31% to 90% of TRL in the well-watered condition. With drought, 100% of TRL was seminal, regardless of line because nodal roots were almost always inhibited in drying topsoil. Irrigation stimulated nodal roots depending on genotype. Shoot size and tillers correlated positively with roots with irrigation, but partitioning depended on genotype and was plastic with drought. Adult root systems of B. distachyon have genetic variation to exploit to increase cereal yields through genes associated with partitioning among roots and their responsiveness to irrigation. Whole-plant phenotypes could enhance gain for droughted environments because root and shoot traits are coselected.Adult plant root systems are relevant to the size and efficiency of seed yield. They supply water and nutrients for the plant to acquire biomass, which is positively correlated to the harvest index (allocation to seed grain), and the stages of flowering and grain development. Modeling in wheat (Triticum aestivum) suggested that an extra 10 mm of water absorbed by such adult root systems during grain filling resulted in an increase of approximately 500 kg grain ha⁻¹ (Manschadi et al., 2006). This was 25% above the average annual yield of wheat in rain-fed environments of Australia. This number was remarkably close to experimental data obtained in the field in Australia (Kirkegaard et al., 2007). Together, these modeling and field experiments have shown that adult root systems are critical for water absorption and grain yield in cereals, such as wheat, emphasizing the importance of characterizing adult root systems to identify phenotypes for productivity improvements.Most root phenotypes, however, have been described for seedling roots. Seedling roots are essential for plant establishment, and hence, the plant’s potential to set seed. For technical reasons, seedlings are more often screened than adult plants because of the ease of handling smaller plants and the high throughput. Seedling-stage phenotyping may also improve overall reproducibility of results because often, growth media are soil free. Seedling soil-free root phenotyping conditions are well suited to dissecting fine and sensitive mechanisms, such as lateral root initiation (Casimiro et al., 2003; Péret et al., 2009a, 2009b). A number of genes underlying root processes have been identified or characterized using seedlings, notably with the dicotyledonous models Arabidopsis (Arabidopsis thaliana; Mouchel et al., 2004; Fitz Gerald et al., 2006; Yokawa et al., 2013) and Medicago truncatula (Laffont et al., 2010) and the cereals maize (Zea mays; Hochholdinger et al., 2001) and rice (Oryza sativa; Inukai et al., 2005; Kitomi et al., 2008).Extrapolation from seedling to adult root systems presents major questions (Hochholdinger and Zimmermann, 2008; Chochois et al., 2012; Rich and Watt, 2013). Are phenotypes in seedling roots present in adult roots given developmental events associated with aging? Is expression of phenotypes correlated in seedling and adult roots if time compounds effects of growth rates and growth conditions on roots? Watt et al. (2013) showed in wheat seedlings that root traits in the laboratory and field correlated positively but that neither correlated with adult root traits in the field. Factors between seedling and adult roots seemed to be differences in developmental stage and the time that growing roots experience the environment.Seedling and adult root differences may be larger in grasses than dicotyledons. Grass root systems have two developmental components: seed-borne (seminal) roots, of which a number emerge at germination and continue to grow and branch throughout the plant life, and stem-borne (nodal or adventitious) roots, which emerge from around the three-leaf stage and continue to emerge, grow, and branch throughout the plant life. Phenotypes and traits of adult root systems of grasses, which include the major cereal crops wheat, rice, and maize, are difficult to predict in seedling screens and ideally identified from adult root systems first (Gamuyao et al., 2012).Phenotyping of adult roots is possible in the field using trenches (Maeght et al., 2013) or coring (Wasson et al., 2014). A portion of the root system is captured with these methods. Alternatively, entire adult root systems can be contained within pots dug into the ground before sowing. These need to be large; field wheat roots, for example, can reach depths greater than 1.5 m depending on genotype and environment. This method prevents root-root interactions that occur under normal field sowing of a plant canopy and is also a compromise.A solution to the problem of phenotyping adult cereal root systems is a model for monocotyledon grasses: Brachypodium distachyon. B. distachyon is a small-stature grass with a small genome that is fully sequenced (Vogel et al., 2010). It has molecular tools equivalent to those available in Arabidopsis (Draper et al., 2001; Brkljacic et al., 2011; Mur et al., 2011). The root system of B. distachyon reference line Bd21 is more similar to wheat than other model and crop grasses (Watt et al., 2009). It has a seed-borne primary seminal root (PSR) that emerges from the embryo at seed germination and multiple stem-borne coleoptile node axile roots (CNRs) and leaf node axile roots (LNRs), also known as crown roots or adventitious roots, that emerge at about three leaves through to grain development. Branch roots emerge from all root types. There are no known anatomical differences between root types of wheat and B. distachyon (Watt et al., 2009). In a recent study, we report postflowering root growth in B. distachyon line Bd21-3, showing that this model can be used to answer questions relevant to the adult root systems of grasses (Chochois et al., 2012).In this study, we used B. distachyon to identify adult plant phenotypes related to the partitioning among seed-borne and stem-borne shoots and roots for the genetic improvement of well-watered and droughted cereals (Fig. 1; Krassovsky, 1926; Navara et al., 1994), nitrogen, phosphorus (Tennant, 1976; Brady et al., 1995), oxygen (Wiengweera and Greenway, 2004), soil hardness (Acuna et al., 2007), and microorganisms (Sivasithamparam et al., 1978). Of note is the study by Krassovsky (1926), which was the first, to our knowledge, to show differences in function related to water. Krassovsky (1926) showed that seminal roots of wheat absorbed almost 2 times the water as nodal roots per unit dry weight but that nodal roots absorbed a more diluted nutrient solution than seminal roots. Krassovsky (1926) also showed by removing seminal or nodal roots as they emerged that “seminal roots serve the main stem, while nodal roots serve the tillers” (Krassovsky, 1926). Volkmar (1997) showed, more recently, in wheat that nodal and seminal roots may sense and respond to drought differently. In millet (Pennisetum glaucum) and sorghum (Sorghum bicolor), Rostamza et al. (2013) found that millet was able to grow nodal roots in a dryer soil than sorghum, possibly because of shoot and root vigor.Open in a separate windowFigure 1.B. distachyon plant scanned at the fourth leaf stage, with the root and shoot phenotypes studied indicated. Supplemental Table S1.

Importin-α-Mediated Nucleolar Localization of Potato Mop-Top Virus TRIPLE GENE BLOCK1 (TGB1) Protein Facilitates Virus Systemic Movement,Whereas TGB1 Self-Interaction Is Required for Cell-to-Cell Movement in Nicotiana benthamiana

Recently, it has become evident that nucleolar passage of movement proteins occurs commonly in a number of plant RNA viruses that replicate in the cytoplasm. Systemic movement of Potato mop-top virus (PMTV) involves two viral transport forms represented by a complex of viral RNA and TRIPLE GENE BLOCK1 (TGB1) movement protein and by polar virions that contain the minor coat protein and TGB1 attached to one extremity. The integrity of polar virions ensures the efficient movement of RNA-CP, which encodes the virus coat protein. Here, we report the involvement of nuclear transport receptors belonging to the importin-α family in nucleolar accumulation of the PMTV TGB1 protein and, subsequently, in the systemic movement of the virus. Virus-induced gene silencing of two importin-α paralogs in Nicotiana benthamiana resulted in significant reduction of TGB1 accumulation in the nucleus, decreasing the accumulation of the virus progeny in upper leaves and the loss of systemic movement of RNA-CP. PMTV TGB1 interacted with importin-α in N. benthamiana, which was detected by bimolecular fluorescence complementation in the nucleoplasm and nucleolus. The interaction was mediated by two nucleolar localization signals identified by bioinformatics and mutagenesis in the TGB1 amino-terminal domain. Our results showed that while TGB1 self-interaction is needed for cell-to-cell movement, importin-α-mediated nucleolar targeting of TGB1 is an essential step in establishing the efficient systemic infection of the entire plant. These results enabled the identification of two separate domains in TGB1: an internal domain required for TGB1 self-interaction and cell-to-cell movement and the amino-terminal domain required for importin-α interaction in plants, nucleolar targeting, and long-distance movement.Pomoviruses are causal agents of important diseases affecting potato (Solanum tuberosum), sugar beet (Beta vulgaris), and bean (Phaseolus vulgaris). Potato mop-top virus (PMTV), the type member of the genus Pomovirus, causes an economically important disease of potato called spraing, inducing brown lines and arcs internally and on the surface of tubers. PMTV is transmitted by the root- and tuber-infecting plasmodiophorid Spongospora subterranea (Jones and Harrison, 1969; Arif et al., 1995).The pomovirus genome is divided into three single-stranded RNA (ssRNA) segments of positive polarity. RNA-Rep encodes the putative RNA-dependent RNA polymerase, the replicase of the virus (Savenkov et al., 1999). RNA-CP encodes a coat protein (CP) and another protein called CP-RT or minor CP, which is produced by translational read-through of the CP stop codon (Sandgren et al., 2001). Whereas CP is the major structural protein of the virions, CP-RT is incorporated in one of the termini of the virus particles and a domain within the read-through region of the protein is needed for transmission of the virus by its vector (Reavy et al., 1998). Moreover, CP-RT, but not CP, interacts with the major movement protein TRIPLE GENE BLOCK1 (TGB1; Torrance et al., 2009), which is encoded by RNA-TGB. Besides encoding a triple gene block of movement proteins, TGB1, TGB2, and TGB3 (Zamyatnin et al., 2004), RNA-TGB also encodes a viral suppressor of RNA silencing, the 8K protein (Lukhovitskaya et al., 2013b).To establish a successful infection in the entire plant, viruses must be able to replicate and to move their genomic components between cells, tissues, and organs. Recently, it has become evident that PMTV utilizes a sophisticated mode of cell-to-cell and long-distance movement that involves two virus transport forms, one represented by the viral nucleoprotein complexes (vRNPs) consisting of virus RNA and the TGB1 protein and another represented by the polar virions containing CP-RT and TGB1 proteins attached to one extremity of virus particles (Torrance et al., 2009; for review, see Solovyev and Savenkov, 2014). Proteins implicated in PMTV cell-to-cell movement include TGB1, TGB2, and TGB3 (Zamyatnin et al., 2004; Haupt et al., 2005a). Indirect evidence suggests that CP-RT is required for the efficient systemic movement of intact virions through its interaction with TGB1 (Torrance et al., 2009).Early in infection, the vRNP is transported on the endoplasmic reticulum actomyosin network and targeted to plasmodesmata by TGB2 and TGB3. Later in infection, fluorescently labeled TGB1 is seen in the nucleus and accumulates in the nucleolus. Nucleolar TGB1 association has been shown to be necessary for long-distance movement (Wright et al., 2010).Two structurally distinct subdomains have been identified in the N terminus of TGB1 proteins of hordeiviruses and pomoviruses (Makarov et al., 2009), an N-terminal domain (NTD) comprising approximately 125 amino acids in PMTV (ssRNA in noncooperative and cooperative manners, respectively. The C-terminal half of TGB1 contains a nucleoside triphosphatase/helicase domain that displays cooperative RNA binding. Previously, Wright et al. (2010) reported that TGB1 expressed from a 35S promoter localizes in the cytoplasm and accumulates in the nucleus and nucleolus with occasional labeling of microtubules (MTs). The MT labeling was apparent behind the leading edge of infection when yellow fluorescent protein (YFP)-TGB1 was expressed from an infectious clone. Deletion of 84 amino acids from the N terminus of TGB1 (representing most of the NTD) resulted in the absence of MTs, and nucleolar labeling and fusion of these 84 N-terminal amino acids to GFP resulted in nucleolar enrichment of GFP but no labeling of MTs. Deletion of the 5′ proximal part of the TGB1 open reading frame (ORF), encoding this N-terminal 84 amino acids, in the virus clone abolished systemic but not cell-to-cell movement. However, such deletion had no effect on TGB1 interactions with the CP-RT or self-interaction (Wright et al., 2010).

Table I.

Structural features of the PMTV TGB1 proteinPositively charged amino acids are set in boldface type and underscored. NoD, Nucleolar localization sequence detector; NS, not shown.

Rethinking Guard Cell Metabolism

Stomata control gaseous fluxes between the internal leaf air spaces and the external atmosphere and, therefore, play a pivotal role in regulating CO₂ uptake for photosynthesis as well as water loss through transpiration. Guard cells, which flank the stomata, undergo adjustments in volume, resulting in changes in pore aperture. Stomatal opening is mediated by the complex regulation of ion transport and solute biosynthesis. Ion transport is exceptionally well understood, whereas our knowledge of guard cell metabolism remains limited, despite several decades of research. In this review, we evaluate the current literature on metabolism in guard cells, particularly the roles of starch, sucrose, and malate. We explore the possible origins of sucrose, including guard cell photosynthesis, and discuss new evidence that points to multiple processes and plasticity in guard cell metabolism that enable these cells to function effectively to maintain optimal stomatal aperture. We also discuss the new tools, techniques, and approaches available for further exploring and potentially manipulating guard cell metabolism to improve plant water use and productivity.Stomata are microscopic, adjustable pores on the leaf surface. The evolution of stomata more than 400 million years ago (Edwards et al., 1986, 1992, 1998) helped facilitate the adaptation of plants to a terrestrial environment, where water is typically a limiting resource. Each stoma is composed of two kidney- or dumbbell-shaped guard cells, whose volume changes to adjust pore aperture, allowing plants to simultaneously regulate CO₂ uptake and water loss. This facilitation of gas exchange by stomatal opening is one of the most essential processes in plant photosynthesis and transpiration, affecting plant water use efficiency and agricultural crop yields (Lawson and Blatt, 2014).Plant physiologists have a long history of investigating the behavior of these fascinating structures, reaching back more than a century to the pioneering work of Sir Francis Darwin (Darwin, 1916) and the American botanist Francis Ernest Lloyd (Lloyd, 1908). Major contributions to stomatal research arose from inventing and improving equipment and methods for quantitatively measuring the effects of environmental factors on stomatal pore aperture. After Darwin’s work, it became clear that the stomatal aperture actively responds to changes in the environment and regulates leaf transpiration rates (Meidner, 1987). Over the past century, much has been learned about their structure, development, and physiology.Despite the anatomical simplicity of the stomatal valve, the surrounding guard cells are highly specialized. Guard cells are morphologically distinct from general epidermal cells and possess complex signal transduction networks, elevated membrane ion transport capacity, and modified metabolic pathways. These features allow rapid modulations in guard cell turgor in response to endogenous and environmental signals, promoting the opening and closure of the stomatal pore in time scales of seconds to hours (Assmann and Wang, 2001). A variety of osmotically active solutes contribute to the buildup of stomatal turgor. Potassium (K⁺) and chloride (Cl⁻) act as the main inorganic ions, and malate²⁻ and sucrose (Suc) function as the main organic solutes. Whereas K⁺ and Cl⁻ are taken up from the apoplast, Suc and malate²⁻ can be imported or synthesized internally using carbon skeletons deriving from starch degradation and/or CO₂ fixation in the guard cell chloroplast (Roelfsema and Hedrich, 2005; Vavasseur and Raghavendra, 2005; Lawson, 2009; Kollist et al., 2014). The accumulation of these osmotica lowers the water potential, promoting the inflow of water, the swelling of guard cells, and the opening of the stomatal pore. Most of the ions taken up, or synthesized by guard cells, are sequestered into the vacuole (Barbier-Brygoo et al., 2011). As a result, the guard cell vacuoles undergo dynamic changes in volume and structure, which are crucial for achieving the full amplitude of stomatal movements (Gao et al., 2005; Tanaka et al., 2007; Andrés et al., 2014). During stomatal closure, guard cells reduce their volume through the release of ions into the cell wall and the consequent efflux of water.The transport of osmolytes across the plasma and tonoplast guard cell membranes is energized by H⁺-ATPase activity, which generates a proton motive force by translocating H⁺ ions against their concentration gradient (Blatt, 1987a, 1987b; Thiel et al., 1992; Roelfsema and Hedrich, 2005; Gaxiola et al., 2007). After the pioneering work of Fischer demonstrated the importance of K⁺ uptake in stomatal opening (Fischer, 1968; Fischer and Hsiao, 1968), K⁺ transport became of central interest and has long been considered the essence of stomatal movement regulation. The development of the voltage clamp technique, along with the relative easy acquisition of knockout mutants and transgenics in the model plant Arabidopsis (Arabidopsis thaliana), helped to uncover the precise mechanism and function of K⁺ fluxes in guard cells. It is well established that changes in membrane potential in response to several stimuli (e.g. light/darkness, CO₂, and abscisic acid [ABA]) alter the direction of K⁺ transport (Thiel et al., 1992; Blatt, 2000; Roelfsema et al., 2001, 2002, 2004). During stomatal opening, the activation of the proton pump generates a sufficiently negative electric potential to cause the uptake of K⁺ through the inward-rectifying K⁺ channels (K⁺_in; Fig. 1). During stomatal closure, K⁺ outflow from outward-rectifying K⁺ channels (K⁺_out) results from membrane depolarization (Fig. 2; Blatt, 1988; Schroeder, 1988; Anderson et al., 1992; Sentenac et al., 1992). Besides being gated by opposing changes in voltage, the activation of (K⁺_out) channels is dependent on the extracellular K⁺ concentration, while that of K⁺_in is not (Blatt, 1988, 1992; Roelfsema and Prins, 1997; Dreyer and Blatt, 2009). There is also strong evidence for H⁺-coupled K⁺ symport in guard cells, which could account for up to 50% of total K⁺ uptake during stomatal opening (Blatt and Clint, 1989; Clint and Blatt, 1989; Hills et al., 2012). At least for K⁺_in, the loss of a single-channel gene in Arabidopsis has little or no impact on stomatal movement (Szyroki et al., 2001), showing the redundancy among the different K⁺_in isoforms and of K⁺ transport in general.Open in a separate windowFigure 1.Integration of guard cell carbohydrate metabolism with membrane ion transport during stomatal opening. Sugars in guard cells can be imported from the apoplast, derive from starch breakdown, or be synthesized in the Calvin cycle. These sugars then can be stored as osmotically active solutes in the vacuole or metabolized in the cytosol to yield energy, reducing equivalents, and phosphoenolpyruvate (PEP). PEP can be further metabolized to pyruvate in the mitochondrial tricarboxylic acid (CAC) cycle or used as carbon skeletons for the biosynthesis of malate via PEP carboxylase (PEPC) and NAD-dependent malate dehydrogenase (NAD-MDH). Malate (which also can be imported from the apoplast) and the inorganic ions K⁺ and Cl⁻ accumulate in the vacuole, lowering the guard cell osmotic potential, thereby promoting stomatal opening. ABCB14, ATP-binding cassette transporter B14; AcetylCoA, acetyl-CoA; ALMT, aluminum-activated malate transporter; ATP-PFK, ATP-dependent phosphofructokinase; AttDT, dicarboxylate transporter; cINV, cytosolic invertase; cwINV, cell wall invertase; Fru6P, Fru-6-P; Fru1,6P₂, fructose 1,6-bisphosphate; Gl6P, Glc-6-P; G3P, glyceraldehyde 3-phosphate; iPGAM, phosphoglycerate mutase isoforms; NRGA1, negative regulator of guard cell ABA signaling1; OAA, oxaloacetate; 2-PGA, 2-phosphoglycerate; 3-PGA, 3-phosphoglycerate; PPi-PFK, PPi-dependent Fru-6-P phosphotransferase; STP, monosaccharide/H⁺ cotransporter; SUC, Suc/H⁺ cotransporter; SuSy, Suc synthase; TPT, triose phosphate/phosphate translocator. Compartments are not to scale. The dotted line indicates multiple metabolic steps.Open in a separate windowFigure 2.Proposed pathways of osmolyte dissipation during stomatal closure. While the removal of Cl⁻ and K⁺ is well described in the literature, the fate of Suc and malate during stomatal closure is unclear. Suc can be cleaved by cytosolic invertase (cINV), and the resulting hexoses can be imported into the chloroplast in the form of Glc-6-P (Glc6P). Glc6P is used subsequently for starch biosynthesis. Malate can be removed from the cell via decarboxylation to pyruvate by malic enzyme (ME) and the subsequent complete oxidation in the mitochondrial tricarboxylic acid (CAC) cycle. Alternatively, malate can be converted to PEP via NAD⁺-dependent malate dehydrogenase (NAD-MDH) and PEP carboxykinase (PEPCK). Gluconeogenic conversion of PEP to Glc6P establishes a possible link between malate removal and starch synthesis. Compartments are not to scale. PEP, Phosphoenolpyruvate; OAA; oxaloacetate; STP, monosaccharide/H⁺ cotransporter; SUC, Suc/H⁺ cotransporter; SuSy, Suc synthase; cINV, cytosolic invertase; NRGA1, negative regulator of guard cell ABA signaling1; ALMT, aluminum-activated malate transporter; GPT, Glc-6-P/Pi translocator; cwINV, cell wall invertase; HK, hexokinase; QUAC1, quickly activating anion channel1.Despite the undisputed importance of K⁺ uptake in stomatal opening, the accumulation of K⁺ ions alone cannot account for the increase in osmotic pressure necessary to explain stomatal aperture. Studies from the 1980s by MacRobbie and Fischer demonstrated that Vicia faba guard cells take up approximately 2 pmol of K⁺ during stomatal opening. Assuming that K⁺ uptake is balanced by the accumulation of similar amounts of counter ions (Cl⁻ and/or malate²⁻), the expected increase in stomatal turgor to approximately 3 MPa is less than the 4.5 MPa expected for fully open stomata (Fischer, 1972; MacRobbie and Lettau, 1980a, 1980b; Chen et al., 2012). The realization that other solutes must accumulate in addition to K⁺ salts was one of the major paradigm shifts in stomatal physiology research in the last decades, equal to the discovery of ion channels. Suc was put forward as the most likely candidate for the additional osmoticum to support stomatal opening (MacRobbie, 1987; Tallman and Zeiger, 1988; Talbott and Zeiger, 1993, 1998). Nonetheless, this research area subsequently failed to attract notice commensurate with its importance.In the last few years, the metabolism of starch, sugars and, organic acids in guard cells has seen a rebirth, making this the perfect time to review the developments in this field. In this review, we focus on photosynthetic carbon assimilation and respiratory metabolism in guard cells and provide a historical overview of the subject that highlights the most up-to-date and novel discoveries in guard cell research. We describe the various metabolic pathways separately, but as metabolism is an integrated network, we also discuss their reciprocal and beneficial interactions. Finally, we highlight their connection with the metabolism in the subjacent mesophyll cells and how they integrate with guard cell signal transduction networks and membrane ion transport to regulate stomatal movements. The enzymes and transporters discussed in this review are listed in

Plastid Genotyping Reveals the Uniformity of Cytoplasmic Male Sterile-T Maize Cytoplasms

Cytoplasmic male-sterile (CMS) lines in maize (Zea mays) have been classified by their response to specific restorer genes into three categories: cms-C, cms-S, and cms-T. A mitochondrial genome representing each of the CMS cytotypes has been sequenced, and male sterility in the cms-S and cms-T cytotypes is linked to chimeric mitochondrial genes. To identify markers for plastid genotyping, we sequenced the plastid genomes of three fertile maize lines (B37, B73, and A188) and the B37 cms-C, cms-S, and cms-T cytoplasmic substitution lines. We found that the plastid genomes of B37 and B73 lines are identical. Furthermore, the fertile and CMS plastid genomes are conserved, differing only by zero to three single-nucleotide polymorphisms (SNPs) in coding regions and by eight to 22 SNPs and 10 to 21 short insertions/deletions in noncoding regions. To gain insight into the origin and transmission of the cms-T trait, we identified three SNPs unique to the cms-T plastids and tested the three diagnostic SNPs in 27 cms-T lines, representing the HA, I, Q, RS, and T male-sterile cytoplasms. We report that each of the tested 27 cms-T group accessions have the same three diagnostic plastid SNPs, indicating a single origin and maternal cotransmission of the cms-T mitochondria and plastids to the seed progeny. Our data exclude exceptional pollen transmission of organelles or multiple horizontal gene transfer events as the source of the mitochondrial urf13-T (unidentified reading frame encoding 13-kD cms-T protein) gene in the cms-T cytoplasms. Plastid genotyping enables a reassessment of the evolutionary relationships of cytoplasms in cultivated maize.Cytoplasmic male sterility has been described in many flowering plant species and is linked to mitochondrial genes encoding toxic proteins. Cytoplasmic male-sterile (CMS) proteins are typically encoded by a chimeric mitochondrial gene assembled from rearranged mitochondrial DNA sequences and contain a hydrophobic, membrane-spanning domain (Hanson and Bentolila, 2004; Chase, 2007; Carlsson et al., 2008; Kubo and Newton, 2008; Chen and Liu, 2014). CMS cytoplasms in maize (Zea mays) are well characterized. Thirty-eight sources of cytoplasmic male sterility have been examined for fertility restoration in 28 inbred backgrounds and classified by their response to specific restorer genes into the cms-C, cms-S, and cms-T groups. The cms-T group is composed of the earlier identified HA, I, Q, RS, and T cytoplasms, which all respond to the same Restorers of fertility nuclear genes Rf1 and Rf2. Plants with the cms-T cytotype are susceptible to Bipolaris (Helminthosporium) maydis race T, a fungal pathogen that causes southern corn leaf blight (Beckett, 1971; Gracen and Grogan, 1974). A distinct mitochondrial genome representing each of the CMS cytotypes has been sequenced (Allen et al., 2007), with male sterility linked to the urf13-T (unidentified reading frame encoding 13-kD cms-T protein) gene in cms-T (Dewey et al., 1987) and the cotranscribed open reading frames (ORFs) orf355/orf77 in cms-S (Zabala et al., 1997). The 13-kD maize mitochondrial protein encoded by the urf13-T gene was shown to confer sensitivity to the T-toxin produced by B. maydis in Escherichia coli, firmly establishing the linkage between the T-type cytoplasmic male sterility and T-toxin sensitivity (Dewey et al., 1988). A collection of CMS lines is searchable at the Maize Genetics and Genomics Database (MaizeGDB) Web site (Sen et al., 2009), and seed may be obtained upon request. We used this resource to learn whether the independently isolated cms-T cytoplasms in cultivated maize are related. In maize, plastids and mitochondria are transmitted to the seed progeny from the maternal parent (Conde et al., 1979). However, maize yields hybrids with relatively distant wild relatives such as Zea luxurians, Zea diploperennis, and Zea perennis (Allen, 2005), and when doing so, it is possible that the mode of organelle inheritance may change to biparental with an increased frequency of organelle leakage via pollen. In an extreme case of an interspecies hybrid, a shift from maternal to paternal inheritance was documented (Hansen et al., 2007). Lineages arising outside a strict maternal mode of organelle inheritance in the cms-T cytoplasm can be reconstructed by analyzing plastid types in the cms-T collection. This necessitated a search for plastid markers.Earlier work yielded very few markers that would be useful for plastid genotyping in cultivated maize (Pring and Levings, 1978). Complete plastid genome sequences are convenient sources of plastid DNA (ptDNA) markers. Prior to our study, the only annotated maize ptDNA sequence in GenBank ({"type":"entrez-nucleotide","attrs":{"text":"NC_001666","term_id":"11994090","term_text":"NC_001666"}}NC_001666; Maier et al., 1995) was assembled from sequencing clones of two maize hybrids (see “Discussion”). To provide markers specific to the lines used in this study, we sequenced the plastid genomes of three fertile lines: A188 representing cytotype NA and B37 representing cytotype NB (Clifton et al., 2004; Allen et al., 2007). The third fertile line, B73, was chosen because its nuclear genome has been sequenced (Schnable et al., 2009). We also sequenced the plastid genome of cms-C, cms-S, and cms-T lines in the B37 nuclear background (B37C, B37S, and B37T lines), in which the mitochondrial genome sequence has been determined (Clifton et al., 2004; Allen et al., 2007). The maize lines with sequenced plastid and mitochondrial genomes are listed in LineCytotypeptDNA Accession No.NucleotidesptDNA ReferenceMitochondrial DNA Accession No.Mitochondrial DNA ReferenceA188NA{"type":"entrez-nucleotide","attrs":{"text":"KF241980","term_id":"540067282","term_text":"KF241980"}}KF241980140,437This study{"type":"entrez-nucleotide","attrs":{"text":"DQ490952","term_id":"93116077","term_text":"DQ490952"}}DQ490952Allen et al. (2007)B73NB{"type":"entrez-nucleotide","attrs":{"text":"KF241981","term_id":"540067367","term_text":"KF241981"}}KF241981140,447This studyB37NNB{"type":"entrez-nucleotide","attrs":{"text":"KP966114","term_id":"893640899","term_text":"KP966114"}}KP966114140,447This study{"type":"entrez-nucleotide","attrs":{"text":"AY506529.1","term_id":"40794996","term_text":"AY506529.1"}}AY506529.1Clifton et al. (2004)B37Ccms-C{"type":"entrez-nucleotide","attrs":{"text":"KP966115","term_id":"893640984","term_text":"KP966115"}}KP966115140,457This study{"type":"entrez-nucleotide","attrs":{"text":"DQ645536","term_id":"102579626","term_text":"DQ645536"}}DQ645536Allen et al. (2007)B37Scms-S{"type":"entrez-nucleotide","attrs":{"text":"KP966116","term_id":"893641069","term_text":"KP966116"}}KP966116140,534This study{"type":"entrez-nucleotide","attrs":{"text":"DQ490951","term_id":"119710194","term_text":"DQ490951"}}DQ490951Allen et al. (2007)B37Tcms-T{"type":"entrez-nucleotide","attrs":{"text":"KP966117","term_id":"893641154","term_text":"KP966117"}}KP966117140,479This study{"type":"entrez-nucleotide","attrs":{"text":"DQ490953","term_id":"93116122","term_text":"DQ490953"}}DQ490953Allen et al. (2007)Open in a separate windowAn alignment of the completed plastid genome sequences facilitated the identification of three single-nucleotide polymorphisms (SNPs) that are unique to the cms-T plastid haplotype. We report here that each of the tested 27 cms-T accessions has the same three diagnostic plastid SNPs, indicating a single origin. Our data exclude exceptional transmission of organelles by pollen or independent horizontal transfer of the urf13-T gene to fertile mitochondrial genomes during domestication as the source of the urf13-T gene in the cms-T cytoplasm.     

Oxidation of P700 in Photosystem I Is Essential for the Growth of Cyanobacteria

The photoinhibition of photosystem I (PSI) is lethal to oxygenic phototrophs. Nevertheless, it is unclear how photodamage occurs or how oxygenic phototrophs prevent it. Here, we provide evidence that keeping P700 (the reaction center chlorophyll in PSI) oxidized protects PSI. Previous studies have suggested that PSI photoinhibition does not occur in the two model cyanobacteria, Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942, when photosynthetic CO₂ fixation was suppressed under low CO₂ partial pressure even in mutants deficient in flavodiiron protein (FLV), which mediates alternative electron flow. The lack of FLV in Synechococcus sp. PCC 7002 (S. 7002), however, is linked directly to reduced growth and PSI photodamage under CO₂-limiting conditions. Unlike Synechocystis sp. PCC 6803 and S. elongatus PCC 7942, S. 7002 reduced P700 during CO₂-limited illumination in the absence of FLV, resulting in decreases in both PSI and photosynthetic activities. Even at normal air CO₂ concentration, the growth of S. 7002 mutant was retarded relative to that of the wild type. Therefore, P700 oxidation is essential for protecting PSI against photoinhibition. Here, we present various strategies to alleviate PSI photoinhibition in cyanobacteria.Low CO₂ fixation efficiency in the Calvin-Benson cycle prevents the utilization of NADPH and ATP in photosynthesis and causes these molecules to accumulate, resulting in oxidative photosynthetic cell damage. High light, low temperature, and CO₂ limitation increase NADPH and ATP levels beyond the Calvin-Benson cycle requirements. Electrons and H⁺ accumulate in the photosynthetic electron transport (PET) system. Excess electrons in the PET system trigger oxidative damage to PSI by forming reactive oxygen species (ROS), including the superoxide anion radical (O₂⁻) and singlet oxygen (¹O₂), within PSI and degrading the P700 reaction center chlorophyll (P700; Sonoike, 1996; Sejima et al., 2014; Zivcak et al., 2015a, 2015b; Takagi et al., 2016). PSI repair has been reported to be a very slow process (Kudoh and Sonoike, 2002), and a recent study showed that it took more than 12 d for damaged PSI in wheat (Triticum aestivum) leaves to recover completely (Zivcak et al., 2015b). PSI photoinhibition, therefore, is very detrimental to oxygenic phototroph growth. Nevertheless, PSI photoinhibition is alleviated by keeping P700 oxidized (Sejima et al., 2014).In the PET system of oxygenic phototrophs, P700 oxidation is a physiological response to environmental variations. In C₃ plants, low CO₂ and/or high light intensity induce P700 oxidation in vivo (Klughammer and Schreiber, 1994; Laisk and Oja, 1994; Miyake et al., 2004, 2005). Several molecular mechanisms are proposed for P700 oxidation wherein the PSI acceptor does not limit the PET reaction. First, H⁺ accumulation on the lumenal side of thylakoid membranes lowers reduced plastoquinone (plastoquinol) oxidation rates in the cytochrome (Cyt) b₆/f complex (Kramer et al., 1999). Second, plastidial terminal oxidase and cyanobacterial respiratory terminal oxidases on the thylakoid membranes suppress PSI electron influx by accepting upstream PSI electrons in the PET system. Oxygen is the final electron acceptor (Beardall et al., 2003; Trouillard et al., 2012; Lea-Smith et al., 2013). Finally, plastoquinol accumulation inhibits the Q-cycle turnover in the Cyt b₆/f complex, which suppresses electron flow from the Cyt b₆/f complex to P700. This reaction is called the reduction-induced suppression of electron flow (RISE; Shaku et al., 2016). Overall, these molecular mechanisms contribute to P700 oxidation, thereby preventing PSI photoinhibition and enabling oxygenic phototrophs to thrive. The proton gradient regulation5 (pgr5) mutant of Arabidopsis (Arabidopsis thaliana) cannot keep P700 oxidized and shows PSI photoinhibition under high-light and fluctuating light conditions (Munekage et al., 2002; Suorsa et al., 2012), which shows the importance of the oxidation of P700 for the protection of PSI in plants.Unlike green plants, P700 oxidation mechanisms in cyanobacteria are unclear. It is known that flavodiiron protein (FLV) could contribute to P700 oxidation. Four FLV isozymes (FLV1−FLV4) have been identified in the model cyanobacterium Synechocystis sp. PCC 6803 (S. 6803; Helman et al., 2003). FLV1 and FLV3 (FLV1/3) function as a heterodimer and catalyze the reduction of oxygen to water on the acceptor side of PSI using NAD(P)H as electron donors (Vicente et al., 2002; Helman et al., 2003; Allahverdiyeva et al., 2013). Unlike FLV1/3, FLV2/4 is induced only under low CO₂ (Zhang et al., 2009) and mediates an oxygen-dependent alternative electron flow (AEF; Shimakawa et al., 2015). In S. 6803, FLV-dependent electron fluxes are coupled to photosynthesis and should alleviate electron overaccumulation in PSI (Helman et al., 2003, 2005; Allahverdiyeva et al., 2013; Shimakawa et al., 2015). Therefore, FLV is expected to contribute to P700 oxidation. The lack of FLV1/3 in S. 6803 causes PSI photoinhibition under artificial fluctuating light (Allahverdiyeva et al., 2013). However, under CO₂ limitation (which suppresses photosynthetic CO₂ fixation), deletions of FLV1/3 and FLV2/4 do not cause PSI photoinhibition in S. 6803 (Zhang et al., 2009) or Synechococcus elongatus PCC 7942 (S. 7942; Shaku et al., 2015), possibly because P700 stays oxidized under CO₂ limitation regardless of the existence of FLV (Shaku et al., 2015). These data imply that FLV is not essential to keep P700 oxidized under CO₂ limitation, at least in S. 6803 and S. 7942.In this study, we found that the lack of FLV1/3 leads to growth inhibition under ambient [CO₂] concentration ([CO₂]) in the cyanobacterium Synechococcus sp. PCC 7002 (S. 7002), unlike S. 6803 and S. 7942 (Zhang et al., 2009; Shaku et al., 2015). The S. 7002 genome, like that of S. 7942, includes genes coding for FLV1/3 isozymes but not for FLV2/4 (Fujisawa et al., 2014). The genetic profiles of flv and other genes related to cyanobacterial AEF, including those of S. 7002, S. 6803, and S. 7942, are summarized in P700 to approximately 10% in the flv knockout mutant of S. 7002 but not in those of S. 6803 or S. 7942. We demonstrated that the deletion of FLV in S. 7002 rendered it unable to oxidize P700, resulting in PSI photoinhibition. These findings show that there are different strategies in cyanobacteria to protect PSI against photooxidative damage under CO₂ limitation.

Table I.

Genetic background of AEF in three cyanobacteria species used in this studyGene homology analyses were performed in Cyanobase (http://genome.microbedb.jp/CyanoBase; Fujisawa et al., 2014). cox, aa₃-type cytochrome c oxidase; cyd, cytochrome bd-type quinol oxidase; arto, cytochrome b_o-type quinol oxidase; ndhD, D subunit of NAD(P)H dehydrogenase.

Focus Issue on Roots: The Optimal Lateral Root Branching Density for Maize Depends on Nitrogen and Phosphorus Availability

Observed phenotypic variation in the lateral root branching density (LRBD) in maize (Zea mays) is large (1–41 cm⁻¹ major axis [i.e. brace, crown, seminal, and primary roots]), suggesting that LRBD has varying utility and tradeoffs in specific environments. Using the functional-structural plant model SimRoot, we simulated the three-dimensional development of maize root architectures with varying LRBD and quantified nitrate and phosphorus uptake, root competition, and whole-plant carbon balances in soils varying in the availability of these nutrients. Sparsely spaced (less than 7 branches cm⁻¹), long laterals were optimal for nitrate acquisition, while densely spaced (more than 9 branches cm⁻¹), short laterals were optimal for phosphorus acquisition. The nitrate results are mostly explained by the strong competition between lateral roots for nitrate, which causes increasing LRBD to decrease the uptake per unit root length, while the carbon budgets of the plant do not permit greater total root length (i.e. individual roots in the high-LRBD plants stay shorter). Competition and carbon limitations for growth play less of a role for phosphorus uptake, and consequently increasing LRBD results in greater root length and uptake. We conclude that the optimal LRBD depends on the relative availability of nitrate (a mobile soil resource) and phosphorus (an immobile soil resource) and is greater in environments with greater carbon fixation. The median LRBD reported in several field screens was 6 branches cm⁻¹, suggesting that most genotypes have an LRBD that balances the acquisition of both nutrients. LRBD merits additional investigation as a potential breeding target for greater nutrient acquisition.At least four major classes of plant roots can be distinguished based on the organ from which they originate: namely the seed, the shoot, the hypocotyl/mesocotyl, and other roots (Zobel and Waisel, 2010). The last class is lateral roots, which form in most plants the majority of the root length, but not necessarily of the root weight, as lateral roots have smaller diameter. Lateral roots start with the formation of lateral root primordia, closely behind the root tip of the parent root. These primordia undergo nine distinguishable steps, of which the last step is the emergence from the cortex of the parent root just behind the zone of elongation, usually only a few days after the first cell divisions that lead to their formation (Malamy and Benfey, 1997). However, not all primordia develop into lateral roots; some stay dormant (Dubrovsky et al., 2006), although dormancy of primordia may not occur in maize (Zea mays; Jordan et al., 1993; Ploshchinskaia et al., 2002). The final number of lateral roots is thereby dependent on the rate of primordia formation as well as the percentage of primordia that develop into lateral roots. This process of primordia formation and lateral root emergence is being studied intensively, including the genes that are activated during the different steps and the hormones regulating the process (López-Bucio et al., 2003; Dubrovsky et al., 2006; Osmont et al., 2007; Péret et al., 2009; Lavenus et al., 2013). Significant genotypic variation in the density of lateral roots has been observed, ranging from no lateral roots to 41 roots cm⁻¹ in maize (Trachsel et al., 2010; Lynch, 2013). This suggests that clear tradeoffs exist for the development of lateral roots and that these genotypes have preprogrammed growth patterns that are adaptive to specific environments. While some of the variation for lateral root branching density (LRBD) that has been observed across environments, for example by Trachsel et al. (2010), is constitutive, many genotypes have strong plasticity responses of LRBD to variations in soil fertility (Zhu et al., 2005a; Osmont et al., 2007). Both the nutrient and carbon status of the plant and the local nutrient environment of the (parent) root tip influence LRBD. Many studies have documented these plasticity responses, and others have tried to unravel parts of the sensing and signaling pathways that regulate LRBD. The utility of root proliferation into a nutrient patch has been studied and debated (Robinson et al., 1999; Hodge, 2004), but much less so the utility of having fewer or more branches across the whole root system. Our understanding of the adaptive significance of variation in LRBD among genotypes is thereby limited, with many studies not accounting for relevant tradeoffs. In this study, we integrate several functional aspects of LRBD with respect to nutrient acquisition, root competition, and internal resource costs and quantify these functional aspects using the functional structural plant model SimRoot. SimRoot simulates plant growth with explicit representation of root architecture in three dimensions (Fig. 1; Supplemental Movie S1). The model focuses on the resource acquisition by the root system and carbon fixation by the shoot while estimating the resource utilization and requirements by all the different organs.

Table I.

Minimum, maximum, and median LRBD in different populations phenotyped by various researchers at several locations in the worldLocations are as follows: D, Juelich, Germany; PA, State College, PA; and SA, Alma, South Africa. Some of the experiments included nutrient treatments: LN, low nitrogen availability; and LP, low phosphorus availability. Data were collected by counting the number of roots along a nodal root segment. Data were supplied by the person indicated under source: H.S., H. Schneider; L.Y., L. York; A.Z., A. Zhan; and J.P., J.A. Postma. WiDiv, Wisconsin Diversity panel; IBM, intermated B73 × Mo17; NAM, nested association mapping.

Immunomodulation by Mesenchymal Stem Cells in Veterinary Species

Mesenchymal stem cells (MSC) are adult-derived multipotent stem cells that have been derived from almost every tissue. They are classically defined as spindle-shaped, plastic-adherent cells capable of adipogenic, chondrogenic, and osteogenic differentiation. This capacity for trilineage differentiation has been the foundation for research into the use of MSC to regenerate damaged tissues. Recent studies have shown that MSC interact with cells of the immune system and modulate their function. Although many of the details underlying the mechanisms by which MSC modulate the immune system have been defined for human and rodent (mouse and rat) MSC, much less is known about MSC from other veterinary species. This knowledge gap is particularly important because the clinical use of MSC in veterinary medicine is increasing and far exceeds the use of MSC in human medicine. It is crucial to determine how MSC modulate the immune system for each animal species as well as for MSC derived from any given tissue source. A comparative approach provides a unique translational opportunity to bring novel cell-based therapies to the veterinary market as well as enhance the utility of animal models for human disorders. The current review covers what is currently known about MSC and their immunomodulatory functions in veterinary species, excluding laboratory rodents.Abbreviations: AT, adipose tissue; BM, Bone marrow; CB, umbilical cord blood; CT, umbilical cord tissue; DC, dendritic cell; IDO, indoleamine 2;3-dioxygenase; MSC, mesenchymal stem cells; PGE₂, prostaglandin E2; VEGF, vascular endothelial growth factorMesenchymal stem cells (MSC, alternatively known as mesenchymal stromal cells) were first reported in the literature in 1968.³⁹ MSC are thought to be of pericyte origin (cells that line the vasculature)^21,22 and typically are isolated from highly vascular tissues. In humans and mice, MSC have been isolated from fat, placental tissues (placenta, Wharton jelly, umbilical cord, umbilical cord blood), hair follicles, tendon, synovial membrane, periodontal ligament, and every major organ (brain, spleen, liver, kidney, lung, bone marrow, muscle, thymus, pancreas, skin).^23,121 For most current clinical applications, MSC are isolated from adipose tissue (AT), bone marrow (BM), umbilical cord blood (CB), and umbilical cord tissue (CT; 11,87,99 Clinical trials in human medicine focus on the use of MSC both for their antiinflammatory properties (graft-versus-host disease, irritable bowel syndrome) and their ability to aid in tissue and bone regeneration in combination with growth factors and bone scaffolds (clinicaltrials.gov).¹³¹ For tissue regeneration, the abilities of MSC to differentiate and to secrete mediators and interact with cells of the immune system likely contribute to tissue healing (Figure 1). The current review will not address the specific use of MSC for orthopedic applications and tissue regeneration, although the topic is covered widely in current literature for both human and veterinary medicine.^57,62,90

Table 1.

Tissues from which MSC have been isolated

Focus on Weed Control: Herbicides as Weed Control Agents: State of the Art: I. Weed Control Research and Safener Technology: The Path to Modern Agriculture

The purpose of modern industrial herbicides is to control weeds. The species of weeds that plague crops today are a consequence of the historical past, being related to the history of the evolution of crops and farming practices. Chemical weed control began over a century ago with inorganic compounds and transitioned to the age of organic herbicides. Targeted herbicide research has created a steady stream of successful products. However, safeners have proven to be more difficult to find. Once found, the mode of action of the safener must be determined, partly to help in the discovery of further compounds within the same class. However, mounting regulatory and economic pressure has changed the industry completely, making it harder to find a successful herbicide. Herbicide resistance has also become a major problem, increasing the difficulty of controlling weeds. As a result, the development of new molecules has become a rare event today.Modern industrial herbicide research begins with the analysis and definition of research objectives. A major part of this lies in the definition of economically important weeds in major arable crops (Kraehmer, 2012). Weed associations change slowly over time. It is important, therefore, to foresee such changes. Today’s weed associations result from events in the distant past. They are associated with the history of crops and the evolution of farm management. In Europe and the Americas, some large-acre crops such as winter oilseed rape and spring oilseed rape (canola), both derived from Brassica spp., and soybean (Glycine max) have attained their current importance only within the last 100 years. Other Old World crops, such as cereals, have expanded over a very long time span and were already rather widespread in Neolithic times (Zohary et al., 2012). The dominance of crop species in agricultural habitats only left room for weed species that could adapt to cultivation technologies. Changes in crop management and the global weed infestation have happened in waves. A major early factor in Europe was presumably the grain trade in the Roman period (Erdkamp, 2005). The Romans spread their preferred crops and, unintentionally, associated weed seeds throughout Europe, Asia, and Africa. A second wave of global vegetation change started in the 16th century after the discovery of the Americas. Crops and weeds were distributed globally by agronomists and botanists. Alien species started to spread on all continents. A third phase can be seen in the 19th century with the industrialization of agriculture and the breeding of competitive crop varieties. The analysis of weed spectra in arable fields grew from this historical background. Weeds are plants interfering with the interests of people (Kraehmer and Baur, 2013), which is why they have been controlled by farmers for millennia.Chemical weed control began just about a century ago with a few inorganic compounds, such as sulfuric acid, copper salts, and sodium chlorate (Cremlyn, 1991). The herbicidal activity of 2,4-dichlorophenoxyacetic acid was detected in the 1940s (Troyer, 2001). Büchel et al. (1977) and Cremlyn (1991), Worthington and Hance (1991). Targeted herbicide research began in the 1950s. In the early days, herbicide candidates progressed from screens purely on the basis of their having biology that would satisfy farmers’ requirements. Mode of action (MoA) studies did not play a major role in the chemical industry prior to the 1970s. Analytical tools were developed and the rapid elucidation of plant pathways and in vitro-based screen assays were used from the 1980s onward. However, in the 1990s and beyond, ever-increasing regulatory and economic pressures have changed the situation of the industry completely, and to satisfy the new requirements, selection criteria beyond biological activity have needed to be applied. Herbicide resistance in weeds has developed into a more serious problem that now constrains the application of certain types of herbicides in some markets. Finally, the introduction of crops resistant to cheap herbicides and of glyphosate-resistant soybean, in particular, took value out of the market and resulted in an enormous economic pressure on the herbicide-producing industry. As a result of this changing and more difficult landscape, the development of new molecules is now a rare event.

Table I.

History of chemical weed control innovationsPost, Postemergence application; Pre, preemergence application, based on data from Cremlyn (1991), Worthington and Hance (1991), Büchel et al. (1977), Herbicide Resistance Action Committee (www.hracglobal.com), and others.