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Light micrographs of cross-sections through leaves of E. johnstonii (Eujo). Arrowheads mark centripetal location of chloroplasts in bundle sheath cells. B, Bundle sheath; M, mesophyll. Bars=20 μm.
Source publication
This study investigated whether Euphorbia subgenus Chamaesyce subsection Acutae contains C3–C4 intermediate species utilizing C2 photosynthesis, the process where photorespired CO2 is concentrated into bundle sheath cells. Euphorbia species in subgenus Chamaesyce are generally C4, but three species in subsection Acutae (E. acuta, E. angusta, and E....
Contexts in source publication
Context 1
... of E. angusta, E. acuta, and E. johnstonii are unifacial with single layers of adaxial and abaxial palisade parenchyma ( Figs 4A, B, 5A). Leaves of E. lata and E. mesembryanthemifolia are bifacial with a single layer of adaxial palisade parenchyma and either two layers of abaxial spongy mesophyll (E. ...
Context 2
... than in the BS cells of the other three species (Table 2). Mesophyll chloroplasts are fewer in E. lata and E. mesembryanthemifolia than the other two species and E. lata has the fewest BS mitochondria of the four species (Table 2). The organelle distribution within M and BS cells in E. johnstonii mirrors that of E. acuta at both the light level (Fig. 5) and the TEM level (data not ...
Citations
... Another CA, perhaps CMT416C, may facilitate recapture of CO 2 released during mitochondrial glycine decarboxylation of photorespiration. This role of CMT416C is supported by CMT416C's predicted mitochondrial targeting sequence; by CMT416C's fluorescence-tag localization between C. merolae's mitochondrion and chloroplast; and by the existence of C 2 photosynthesis, a plant carbon-concentrating mechanism which recaptures carbon from mitochondrial glycine decarboxylation (Sage et al. 2011;Rademacher et al. 2017). However, our model (Fig. 5) depicts an alternative function of CMT416C. ...
Cyanidioschyzon merolae is an extremophilic red microalga which grows in low-pH, high-temperature environments. The basis of C. merolae’s environmental resilience is not fully characterized, including whether this alga uses a carbon-concentrating mechanism (CCM). To determine if C. merolae uses a CCM, we measured CO2 uptake parameters using an open-path infra-red gas analyzer and compared them to values expected in the absence of a CCM. These measurements and analysis indicated that C. merolae had the gas-exchange characteristics of a CCM-operating organism: low CO2 compensation point, high affinity for external CO2, and minimized rubisco oxygenation. The biomass δ¹³C of C. merolae was also consistent with a CCM. The apparent presence of a CCM in C. merolae suggests the use of an unusual mechanism for carbon concentration, as C. merolae is thought to lack a pyrenoid and gas-exchange measurements indicated that C. merolae primarily takes up inorganic carbon as carbon dioxide, rather than bicarbonate. We use homology to known CCM components to propose a model of a pH-gradient-based CCM, and we discuss how this CCM can be further investigated.
Supplementary Information
The online version contains supplementary material available at 10.1007/s11120-023-01000-6.
... However, the evidence provided by Cao et al. to support their (correct) conclusion was insufficient relative to the typically accepted standard for characterising the photosynthetic type of a species (e.g. Lundgren et al., 2016;Marshall et al., 2007;Sage et al., 2011). Net photosynthetic rate was measured but only under a single (unspecified) set of conditions rather than across a range of CO 2 concentrations, which means that the CCP cannot be established. ...
... These δ 13 C values are much more negative than those measured in our study. While very negative δ 13 C values can indicate double discrimination against the heavier 13 C isotope as a result of a glycine shuttle (Von Caemmerer, 1992), our anatomy and physiology data are not consistent with Paulownia using C 2 photosynthesis (Lundgren et al., 2016;Sage et al., 2011). Instead, the high level of carbon isotope discrimination identified in Cao et al.'s study is likely linked to the environmental conditions during plant growth, as discrimination is greater given an abundant supply of water and exposure to low photon flux density (Cernusak et al., 2013). ...
Societal Impact Statement
C4 photosynthesis is an ultra‐efficient mode of photosynthesis found in some of our most productive crop species yet is notably rare in trees. Given C4 photosynthesis is associated with high yield in herbaceous species, especially under hot and dry conditions, C4 trees may seem an attractive prospect for biomass production and carbon sequestration in a rapidly changing climate. This may explain why some in the literature have optimistically linked C4 photosynthesis with the exceptionally fast‐growing tree Paulownia. However, this claim is lacking in evidence and represents an example of poor citation practices leading to the spread of misinformation.
Summary
The rapid growth of trees in genus Paulownia (Paulowniaceae) has been attributed in the literature to their use of C4 photosynthesis, a complex trait that confers increased photosynthetic efficiency under certain environmental conditions. After careful examination of citations used to support the idea that Paulownia species use C4 photosynthesis, we find that there is no data underpinning this claim. Despite this, many investment schemes utilise information about the physiology of Paulownia, including photosynthetic type, to legitimise the use of Paulownia trees for financial investment and carbon offsetting.
This study uses leaf physiology, anatomy and stable isotope data to determine whether or not three species in Paulownia (Paulownia tomentosa, Paulownia fortunei and Paulownia kawakamii) use C4 photosynthesis. These data are compared with existing data for C3 and C4 woody species in the literature.
We show that the leaf physiology, anatomy and stable isotope phenotypes of the three Paulownia trees considered in the study are not consistent with those of C4 plants.
Our findings highlight how inaccurate citation of scientific findings can contribute to the spread of misinformation beyond the scientific community, as some of those promoting investments in Paulownia plantations reference the photosynthetic superiority of Paulownia as a means to legitimise its use in carbon offsetting.
... Enzyme activities were assayed at 30 C by coupling the absorption change at 340 nm from the oxidation/reduction of NAD(P)H to the activity of the enzyme examined (Ashton et al. 1990;Keys and Parry, 1990;Sage et al., 2011). For assays of the activities of PEPC, NADP-malic enzyme (NADP-ME), NADP-malate dehydrogenase (NADP-MDH), ribulose 1,5bisphosphate carboxylase/oxygenase (Rubisco), aspartate aminotransferase (Ast-AT), and alanine aminotransferase (Alt-AT), the leaves were frozen in liquid nitrogen and kept in -70 C until assay. ...
Flaveria is a leading model for C4 plant evolution due to the presence of a dozen C3-C4 intermediate species, many of which are associated with a phylogenetic complex centered around F. linearis. To investigate C4 evolution in Flaveria, we updated the Flaveria phylogeny and evaluated gas exchange, starch δ13C, and activity of C4 cycle enzymes in 19 Flaveria species and 28 populations within the F. linearis complex. A principal component analysis identified six functional clusters: i) C3, ii) sub-C2, iii) full C2, iv) enriched C2, v) sub-C4, and vi) fully C4 species. The sub-C2 species lacked a functional C4 cycle, while a gradient was present in the C2 clusters from little to modest C4 cycle activity as indicated by δ13C and enzyme activities. Three Yucatan populations of F. linearis had photosynthetic CO2 compensation points equivalent to C4 plants but showed little evidence for an enhanced C4 cycle, indicating they have an optimized C2 pathway that recaptures all photorespired CO2 in the bundle sheath (BS) tissue. All C2 species had enhanced aspartate aminotransferase activity relative to C3 species and most had enhanced alanine aminotransferase activity. These aminotransferases form aspartate and alanine from glutamate and in doing so help return photorespiratory nitrogen (N) from BS to mesophyll cells, preventing glutamate feedback onto photorespiratory N assimilation. Their use requires upregulation of parts of the C4 metabolic cycle to generate carbon skeletons to sustain N return to the mesophyll, and thus could facilitate the evolution of the full C4 photosynthetic pathway.
... Intermediate C 3 -C 4 lacking Kranz anatomy was observed in other angiosperm groups, as in Flaveria Juss. (Asteraceae; Holaday et al. 1984), Parthenium L. (Asteraceae; Moore et al. 1987), Euphorbia L. (Euphorbiaceae; Sage et al. 2011), Heliotropium L. (Boraginaceae; Muhaidat et al. 2011) and Alternanthera Forssk. (Amaranthaceae; Rajendrudu et al. 1986) and several authors indicated that studies on this issue are important to the comprehension of the evolutionary progression from C 3 to C 4 photosynthesis, including stages of formation of proto-Kranz anatomy (Sage et al. 2012). ...
Portulaca minensis is a poorly known species of Portulacaceae, considered exclusive to the campo rupestre vegetation in Minas Gerais, Brazil. It shares morphological features with members of the Cryptopetala clade, composed of P. mucronata, P. cryptopetala, and P. hirsutissima. The purpose of this paper is to expand the knowledge about the taxonomy, distribution, and micromorphology of P. minensis, comparing it to its morphologically related species occurring in the Espinhaço Range, along Eastern Brazil. We analyzed specimens of this group in herbaria and in the field and gathered data on micromorphology of seeds surface and pollen grains using scanning electron microscopy. In addition, we also analyzed leaf anatomy using light microscopy for the first time to this species. Portulaca minensis can be distinguished from its allies within the P. hirsutissima complex by glabrous leaves and sepaloid (vs. pilose) and from P. mucronata, it differs by reddish sepaloid structures (vs. greenish). As this species showed leaf anatomy with intermediate metabolism C 3-C 4 , we consider it as a probable member of the Cryptopetala clade. The pantocolpate pollen grains were similar to most Portulaca species (except the P. hirsutissima complex), but the slightly convex ornate seeds with par-dome type projections were considered diagnostic to P. minensis. We also indicate new localities of occurrence to P. minensis in the state of Bahia, expanding the species distribution toward the Northeastern region. This species is classified as endangered according to the IUCN criteria (EN B1 + b(i, ii, iii), with EOO = 3,557,978 km 2 and AOO = 28,000 km 2 .
... The activities of PEPC, NADP-malic enzyme, and NADmalic enzyme were assayed at 30 • C using a coupled-enzyme assay that measured oxidation/reduction rate of NADP(H) or NAD(H) at a wavelength of 340 nm using a Hewitt-Packard 8230 spectrophotometer following procedures in Ashton et al. (1990) as modified by Sage T. L. et al. (2011). Two to three cm 2 of recent, fully-mature leaves of A. incarnata, B. coccinea, and N. capitata were sampled under full illumination in the greenhouse and then rapidly ground using a glass tissue homogenizer in an extraction buffer (100 mM HEPES -pH 7.6, 5 mM MgCl 2 , 10 mM KHCO 3 , 2 mM EDTA, 10 mM 6-aminocaproic acid, 2 mM benzamide, 1 mM phenylmethylsulfonyl fluoride, 1% (w/v) PVPP, 2% (w/v) PVP, 0.5% Triton X-100, 2% (w/v) BSA, 5 mM DTT, 1% (w/v) casein). ...
... The structural characteristics observed here are consistent with patterns in other C 4 clades. NADP-ME species among C 4 grasses, sedges and eudicots (as shown in Flaveria, Euphorbia, Gomphrena, Heliotropium, Salsola, and Tribulus) share with Boerhavia and Portulaca pilosa the pattern of enlarged chloroplasts with weakly developed grana stacks, and few BS mitochondria (Carolin et al., 1978;Kim and Fisher, 1990;Ueno, 1996Ueno, , 2013Voznesenskaya et al., 1999;Yoshimura et al., 2004;Muhaidat et al., 2011;Sage T. L. et al., 2011;Lauterbach et al., 2019). NAD-ME eudicots in Amaranthus, Atriplex, Anticharis, Cleome, Gisekia, Salsola, Suaeda, and Tecticornia share with Allionia and P. oleracea the pattern of enlarged chloroplasts with well-developed grana and large numbers of interspersed mitochondria (Carolin et al., 1978;Voznesenskaya et al., 1999Voznesenskaya et al., , 2007Khoshravesh et al., 2012;Bissinger et al., 2014;Oakley et al., 2014). ...
... The activities of PEPC, NADP-malic enzyme, and NADmalic enzyme were assayed at 30 • C using a coupled-enzyme assay that measured oxidation/reduction rate of NADP(H) or NAD(H) at a wavelength of 340 nm using a Hewitt-Packard 8230 spectrophotometer following procedures in Ashton et al. (1990) as modified by Sage T. L. et al. (2011). Two to three cm 2 of recent, fully-mature leaves of A. incarnata, B. coccinea, and N. capitata were sampled under full illumination in the greenhouse and then rapidly ground using a glass tissue homogenizer in an extraction buffer (100 mM HEPES -pH 7.6, 5 mM MgCl 2 , 10 mM KHCO 3 , 2 mM EDTA, 10 mM 6-aminocaproic acid, 2 mM benzamide, 1 mM phenylmethylsulfonyl fluoride, 1% (w/v) PVPP, 2% (w/v) PVP, 0.5% Triton X-100, 2% (w/v) BSA, 5 mM DTT, 1% (w/v) casein). ...
... The structural characteristics observed here are consistent with patterns in other C 4 clades. NADP-ME species among C 4 grasses, sedges and eudicots (as shown in Flaveria, Euphorbia, Gomphrena, Heliotropium, Salsola, and Tribulus) share with Boerhavia and Portulaca pilosa the pattern of enlarged chloroplasts with weakly developed grana stacks, and few BS mitochondria (Carolin et al., 1978;Kim and Fisher, 1990;Ueno, 1996Ueno, , 2013Voznesenskaya et al., 1999;Yoshimura et al., 2004;Muhaidat et al., 2011;Sage T. L. et al., 2011;Lauterbach et al., 2019). NAD-ME eudicots in Amaranthus, Atriplex, Anticharis, Cleome, Gisekia, Salsola, Suaeda, and Tecticornia share with Allionia and P. oleracea the pattern of enlarged chloroplasts with well-developed grana and large numbers of interspersed mitochondria (Carolin et al., 1978;Voznesenskaya et al., 1999Voznesenskaya et al., , 2007Voznesenskaya et al., , 2008Khoshravesh et al., 2012;Bissinger et al., 2014;Oakley et al., 2014). ...
C4 photosynthesis evolved over 65 times, with around 24 origins in the eudicot order Caryophyllales. In the Caryophyllales family Nyctaginaceae, the C4 pathway is known in three genera of the tribe Nyctagineae: Allionia, Okenia and Boerhavia. Phylogenetically, Allionia and Boerhavia/Okenia are separated by three genera whose photosynthetic pathway is uncertain. To clarify the distribution of photosynthetic pathways in the Nyctaginaceae, we surveyed carbon isotope ratios of 159 species of the Nyctaginaceae, along with bundle sheath (BS) cell ultrastructure, leaf gas exchange, and C4 pathway biochemistry in five species from the two C4 clades and closely related C3 genera. All species in Allionia, Okenia and Boerhavia are C4, while no C4 species occur in any other genera of the family, including three that branch between Allionia and Boerhavia. This demonstrates that C4 photosynthesis evolved twice in Nyctaginaceae. Boerhavia species use the NADP-malic enzyme (NADP-ME) subtype of C4 photosynthesis, while Allionia species use the NAD-malic enzyme (NAD-ME) subtype. The BS cells of Allionia have many more mitochondria than the BS of Boerhavia. Bundle sheath mitochondria are closely associated with chloroplasts in Allionia which facilitates CO2 refixation following decarboxylation by mitochondrial NAD-ME. The close relationship between Allionia and Boerhavia could provide insights into why NADP-ME versus NAD-ME subtypes evolve, particularly when coupled to analysis of their respective genomes. As such, the group is an excellent system to dissect the organizational hierarchy of convergent versus divergent traits produced by C4 evolution, enabling us to understand when convergence is favored versus when divergent modifications can result in a common phenotype.
... CAM lineages and C 4 lineages tend to cluster in certain regions of the angiosperm phylogenetic tree (Sage et al., 2011a;Edwards and Ogburn, 2012;Christin et al., 2015), with particularly closely related CAM and C 4 lineages identified within the Aizoaceae, Euphorbiaceae, and Portulacineae (Klak et al., 2003(Klak et al., , 2017Sage et al., 2011b;Horn et al., 2012Horn et al., , 2014Ocampo et al., 2013;Christin et al., 2014). Following the discovery of CAM and C 4 photosynthesis in Euphorbia and Portulaca, Sesuvium becomes only the third genus known to contain both types of carbon-concentrating photosynthesis. ...
... Winter et al., unpublished results). Within the genus Euphorbia, which contains C 3 , C 4 , and CAM species (Webster et al., 1975;Sage et al., 2011b;Yang and Berry, 2011), there are no reports as yet of CAM within the C 4 clade (subg. Chamaesyce), although it appears to have repeatedly evolved alongside succulence from C 3 lineages (Horn et al., 2014). ...
Demonstration of crassulacean acid metabolism (CAM) in species with low usage of this system relative to C3-photosynthetic CO2 assimilation can be challenging experimentally but provides crucial information on the early steps of CAM evolution. Here, weakly expressed CAM was detected in the well-known pantropical coastal, leaf-succulent herb Sesuvium portulacastrum, demonstrating that CAM is present in the Sesuvioideae, the only sub-family of the Aizoaceae in which it had not yet been shown conclusively. In outdoor plots in Panama, leaves and stems of S. portulacastrum consistently exhibited a small degree of nocturnal acidification which, in leaves, increased during the dry season. In potted plants, nocturnal acidification was mainly facultative, as levels of acidification increased in a reversible manner following the imposition of short-term water-stress. In drought-stressed plants, nocturnal net CO2 exchange approached the CO2-compensation point, consistent with low rates of CO2 dark fixation sufficient to eliminate respiratory carbon loss. Detection of low-level CAM in S. portulacastrum adds to the growing number of species that cannot be considered C3 plants sensu stricto, although they obtain CO2 principally via the C3 pathway. Knowledge about the presence/absence of low-level CAM is critical when assessing trajectories of CAM evolution in lineages. The genus Sesuvium is of particular interest because it also contains C4 species.
... From studies of both eudicots and Neurachne, the initial phase of C 2 evolution appears to be the accumulation of organelles in the sheath tissue (BS in Flaveria, Heliotropium, and Euphorbia, MS in Neurachne), to strengthen, or "activate," the photosynthetic potential of the sheath tissue (Muhaidat et al., 2011;Sage et al., 2011bSage et al., , 2013Schulze et al., 2013). In C 3 Flaveria, Euphorbia, and Heliotropium, this activation occurs in tandem with increased BS width. ...
The Australian grass subtribe Neurachninae contains closely related species that use C3, C4, and C2 photosynthesis. To gain insight into the evolution of C4 photosynthesis in grasses, we examined leaf gas exchange, anatomy and ultrastructure, and tissue localization of Gly decarboxylase subunit P (GLDP) in nine Neurachninae species. We identified previously unrecognized variation in leaf structure and physiology within Neurachne that represents varying degrees of C3-C4 intermediacy in the Neurachninae. These include inverse correlations between the apparent photosynthetic carbon dioxide (CO2) compensation point in the absence of day respiration (C
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) and chloroplast and mitochondrial investment in the mestome sheath (MS), where CO2 is concentrated in C2 and C4Neurachne species; width of the MS cells; frequency of plasmodesmata in the MS cell walls adjoining the parenchymatous bundle sheath; and the proportion of leaf GLDP invested in the MS tissue. Less than 12% of the leaf GLDP was allocated to the MS of completely C3 Neurachninae species with C
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values of 56-61 μmol mol-1, whereas two-thirds of leaf GLDP was in the MS of Neurachne lanigera, which exhibits a newly-identified, partial C2 phenotype with C
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of 44 μmol mol-1 Increased investment of GLDP in MS tissue of the C2 species was attributed to more MS mitochondria and less GLDP in mesophyll mitochondria. These results are consistent with a model where C4 evolution in Neurachninae initially occurred via an increase in organelle and GLDP content in MS cells, which generated a sink for photorespired CO2 in MS tissues.
... Paleoecological work produced a wide range of isotope, microfossil, and proxy data which placed C 4 species on landscapes of the past 20 million years, thereby complimenting the molecular clock estimates Koch 2003, 2004;McInerney et al. 2011;Strömberg 2011). Meanwhile, physiological studies identified many new C 3 -C 4 intermediate species, which in tandem with phylogenetic work facilitated comparative approaches to test hypotheses of C 4 evolution (Marshall et al. 2007;Voznesenskaya et al. 2007Voznesenskaya et al. , 2010Voznesenskaya et al. , 2013Muhaidat et al. 2011;Ocampo et al. 2010Ocampo et al. , 2013Khoshravesh et al. 2012;Sage et al. 2011bSage et al. , 2013Lundgren et al. 2016). These advances informed a new generation of models that combined biochemical and evolutionary-landscape theory to predict evolutionary pathways from C 3 to C 4 photosynthesis (Williams et al. 2013;Heckmann et al. 2013;Heckmann 2016). ...
... Next, in the "C 3 enabled" stage (termed the C 3 + stage for short), certain C 3 taxa acquire features which predispose, or enable, the initiation of C 2 evolution. These include duplicated genes, which allow for neofunctionalization of gene copies, more prominent BS cells with more organelles, and reduced distance between veins (Monson 2003;Muhaidat et al. 2011;Christin et al. 2013;Sage et al. 2011bSage et al. , 2013Sage et al. , 2014Williams et al. 2013;Voznesenskaya et al. 2007Voznesenskaya et al. , 2010Voznesenskaya et al. , 2013. In the BS cells of C 3 + species, the organelles are usually positioned along the outer wall opposite the intercellular air spaces, in a pattern typical of C 3 M cells (Muhaidat et al. 2011;Sage et al. 2013Sage et al. , 2014. ...
... Activated BS and close vein spacing are considered enabling traits, because they can facilitate a faster flux of metabolites to the BS, and have greater biochemical capacity to metabolize photorespiratory metabolites which may overflow from the M cells Christin et al. 2013). The C 3 + phenotype has been observed in the eudicot clades Flaveria, Euphorbia, and Heliotropium, and in the PACMAD grasses (Muhaidat et al. 2011;Sage et al. 2011bSage et al. , 2013Christin et al. 2013). Most of these enabled C 3 species are from hot, drought-prone environments, particularly in the eudicot clades. ...
The evolution of C4 photosynthesis requires an intermediate phase where photorespiratory glycine produced in the mesophyll cells must flow to the vascular sheath cells for metabolism by glycine decarboxylase. This glycine flux concentrates photorespired CO2 within the sheath cells, allowing it to be efficiently refixed by sheath Rubisco. A modest C4 biochemical cycle is then upregulated, possibly to support the refixation of photorespired ammonia in sheath cells, with subsequent increases in C4 metabolism providing incremental benefits until an optimized C4 pathway is established. 'Why' C4 photosynthesis evolved is largely explained by ancestral C3 species exploiting photorespiratory CO2 to improve carbon gain and thus enhance fitness. While photorespiration depresses C3 performance, it produces a resource (photorespired CO2) that can be exploited to build an evolutionary bridge to C4 photosynthesis. 'Where' C4 evolved is indicated by the habitat of species branching near C3-to-C4 transitions on phylogenetic trees. Consistent with the photorespiratory bridge hypothesis, transitional species show that the large majority of > 60 C4 lineages arose in hot, dry, and/or saline regions where photorespiratory potential is high. 'When' C4 evolved has been clarified by molecular clock analyses using phylogenetic data, coupled with isotopic signatures from fossils. Nearly all C4 lineages arose after 25 Ma when atmospheric CO2 levels had fallen to near current values. This reduction in CO2, coupled with persistent high temperature at low-to-mid-latitudes, met a precondition where photorespiration was elevated, thus facilitating the evolutionary selection pressure that led to C4 photosynthesis.
... Physiologically, noted that this subgeneric group includes all known variants of photosynthesis, the C 3 , C 4 and CAM pathways and C 2 pathway. This subgenus is widely distributed in the Americas, from Canada to Argentina and has a center of diversity in Mexico and Central America, ranking from tropical forests to deserts and areas of disturbance with altitudes ranging from sea level to 3000 m (Sage et al., 2011;Yang and Berry, 2011;Horn et al., 2012;Yang et al., 2012). ...
The Candelilla plant (Euphorbia antisyphilitica) has been used as a traditional source of hydrocarbons to elaborate candles but recently the potential applications of several compounds has expanded the perspectives for this crop. Phylogenetic analyzes of the Euphorbia genus placed to E. antisyphilitica in the Chamaesyce subgenus, where most of American Euphorbia species are located, including to plants adapted to dry land conditions. In this work a brief anatomical description of aerial stem, rhizome and root is showed and also a comparison of the variable wax compositions is presented to illustrate that exploitation of this resource should consider variations at environmental, genetic and agronomical levels. An integrative knowledge of physiology, biochemistry, genetic improvement, pest and disease studies of this crop would lead to increase yielding for future massive production of Candelilla.
