**1. Introduction**

Thin films of scandium-substituted aluminum nitride (Al1−xScxN or 'AlScN') are a pioneering new class of ferroelectric materials with wurtzite-type structure and unidirectional polarization reversal resulting in square-like hysteresis loops [1–5]. Their ultra-stable remnant polarization and high coercive fields promise ferroelectric field-effect transistorbased non-volatile memory devices maintaining large memory windows combined with high access speed, high endurance and low energy consumption [6]. In this respect, the integration of AlScN on Si has been demonstrated recently for ferroelectric field-effect transistors as well as GaN technology-based structures, e.g., for high electron mobility transistors [7–10] with integrated memory functions. Further, thin films of AlScN are potential candidates for high-temperature actuation and sensing applications in harsh environments (T > 500 ◦C) or for high-temperature non-volatile memory due to remarkable temperature

**Citation:** Wolff, N.; Islam, M.R.; Kirste, L.; Fichtner, S.; Lofink, F.; Žukauskaite, A.; Kienle, L. Al ˙ 1−xScxN Thin Films at High Temperatures: Sc-Dependent Instability and Anomalous Thermal Expansion. *Micromachines* **2022**, *13*, 1282. https://doi.org/10.3390/ mi13081282

Academic Editor: Niall Tait

Received: 22 June 2022 Accepted: 4 August 2022 Published: 8 August 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

stability of the wurtzite-type structure and its piezo/ferroelectric properties at least up to 1100 ◦C [11].

In contrast, many conventional oxide-based piezoelectric and especially ferroelectric thin film materials such as Pb1−xZrxTiO3 or BaTiO3 are limited in their usable temperature range either by phase transitions (relatively low Curie temperatures), which describe the transition from the pyroelectric and thus ferroelectric state to a paraelectric state, or chemical decomposition [12,13]. Numerous detailed studies on temperature-dependent physical properties, e.g., the change in coercive fields [14,15], the pyroelectric coefficient [16], thermo-electro-acoustic coupling and thermal expansion [17–19], have been conducted to date, but only moderate temperatures of max. 400 ◦C were investigated. Recently, we also reported on in situ and ex situ high-temperature investigations, addressing the structural stability of Al1−xScxN (x~0.3) thin films [11,20]. However, depending on the annealing conditions, thin film stack and deposition conditions, both degradation of crystal quality and improvement after annealing were observed. Thereafter, the overall picture of the thermal stability of AlScN thin films in the high-temperature regime remains diffuse and a discussion of the influence of the Sc content is still missing.

Structurally, the substitution of Al atoms by Sc atoms into the AlN4 tetrahedra distorts the (Al, Sc)-N bond lengths and bond angles of the three bonds forming the basal plane of the new tetrahedral *M*N4 (*M* = Al, Sc) unit. As a result, the in-plane lattice parameter *a* and out-of-plane lattice parameter *c* change anisotropically with increasing concentration of Sc [19]. Statistical calculations on supercell models support this relationship showing that Sc increases disorder and symmetry breaking, establishing a tilt of the *M*N4 tetrahedra [21]. In consequence, a pronounced elastic softening along the *c*-axis is observed [17] which couples to the evolution of the internal displacement parameter *u* of the cation–anion distance described by the product *uc*. The compromise between the ideal tetrahedral coordination of Al (*u* = 3/8) and the favored octahedral coordination of Sc (*u* = 1/2) results in the displacement of Sc in the tetrahedra. With increasing Sc concentration, this displacement approaches a theoretical layered hexagonal phase with *u = 0.5* promoting phase transition [21]. The flattened energy landscape with increasing Sc concentration [22] also improves the materials' piezoelectric response up to *d*33~27 pC/N at *x* = 0.43 [23] and promotes ferroelectric switching in high electric fields [1]. In conclusion, the substitution of Al by Sc in AlN in tetrahedral positions destabilizes the wurtzite-type crystal structure, resulting in a phase transformation above *x* > 0.46 to a non-polar cubic AlScN exhibiting rocksalt-type structure with octahedral coordination [24,25].

With this contribution, we extend our previous work on Al0.73Sc0.27N thin films on templates of Mo(110)/AlN(0001)/Si(001) [11] and match our new results to high-temperature data of Al1−xScxN(0001)/Al2O3(0001) to provide a joint perspective on AlScN films subjected to the high-temperature regime. In the latter systems, the Sc content was varied from Sc *x* = 0.0 to 0.40 to study Sc content-dependent thermal expansion behavior and high-temperature stability. The discussed results are based on the combination of X-ray diffraction techniques and in situ annealing experiments. Our measurements reveal the increasing structural destabilization of AlScN with increasing Sc content. Further, the demonstration of thermally activated effects is described, which drive an unexpected volume expansion at intermediate and high temperatures exceeding 550 ◦C. The high-temperature branch of thermal expansion is separated into intrinsic and extrinsic contributions related to oxygen impurities resulting in an irreversible change in the lattice parameters after annealing. Our results could have implications for the integration of AlScN thin films into metal–ferroelectric–metal capacitors for actuator, sensor or computing structures designed for high-temperature operation exceeding 500 ◦C up to the materials' Curie temperature of >1100 ◦C.


**5. miRNAs and cDNA-Microarray Response to Salinity Stress**

Salt intrusion from saline soils and irrigation water is one of the most severe and harmful risks to reduce agricultural production and adverse effects on cultivated land and the geographical distribution of plant species [70,132,133], coupled with oxidative stress [134]. The most imperative cations in saline soils are calcium, potassium, magnesium, and sodium, and the main anions in saline soils are chloride, bicarbonate, sulfate, nitrate, and carbonates. Other electrolytes causative to salinity are borane, molybdenum, strontium, silicon dioxide, aluminum cation, and barium ion [135,136]. Higher concentrations of sodium chloride (NaCl) typically affect plant development, metabolism, and physiology at various metabolic phases (ion toxicity, nutrient imbalance, and oxidative stress) [70,137]. Despite such advances in scientific research, it remains unclear about the underlying molecular mechanism of salinity responses in plants. However, based on the combination of microarray and inhibition subtractive hybridization (SSH), changes in the transcriptome profile caused by salt induction were studied and evaluated [138]. Investigation of complete transcriptomics suggests that these processes, such as the synthesis of osmolytes and ion carriers and the regulation of transcription and translation mechanisms, have distinctive reactions under salinity stress. In particular, the introduction of transcripts of specific TFs, ribosomal genes, RNA-binding proteins, and translation initiation and elongation factors has been testified [139,140].

Using cDNA microarray in *Synechocystis*, 19 genes were reported to be instantaneously regulated under salinity stress. The salt- and osmo-regulated genes, and some putative sensor molecules, have been implicated during salinity stress signaling [35]. Several differentially regulated miRNAs have been reported against salinity stress. In *A. thaliana*, several microRNAs are regulated against salinity stress, such as miR156, miR158, miR159, miR165, miR167, miR168, miR169, miR171, miR319, miR393, miR394, miR396, and miR397 (Table 3, Figure 2) [84]. In *Populus trichocarpa*, miR1445, miR1447, miR1446a-e, miR530a, and miR171l-n were down-regulated (Table 3) [141]. Arenas-Huertero et al. [31] reported, in *Proteus vulgaris*, the production of miRS1 and miR159.2 expression in response to salinity. Furthermore, miR169g and family members of miR169n were induced in saline-rich conditions [142]. However, there is a need to discover and annotate novel functional genes which have a probable function against salinity stress. Subsequently, a large number of genes in plants still have unknown functions [143]. Recent studies revealed that specific down-regulation of the bacterial-type *phosphoenolpyruvate carboxylase* (*PEPC*) gene *Atppc4* by artificial microRNA enhanced the salinity tolerance in *A. thaliana*. The increased salinity tolerance might be linked to enhanced PEPC activity [10,144]. Transcript control for salinitytolerant rice with microarrays, like 1728 cDNAs from salinity-stressed roots libraries, was studied in response to high salinity (Table 3) [144–146].

A tiling path microarray was used to examine the high-throughput expression profiling patterns under various environmental stresses for all of the known miRNAs [16,70] (Tables 1 and 4). The analysis revealed that the effects of miRNAs under low-temperature, drought, and high salinity with miRNA chips represent, approximately, all of the reported miRNAs cloned or recognized in *A. thaliana* (L.). High salinity stress agitates homeostasis in water potential. Extreme changes in water homeostasis and ions lead to molecular breakdown, stunted growth, and even the death of cells or whole plants [16,147].

**Table 4.** Software and tools used for the detection of plant miRNA and cDNA microarray data analysis.

#### **Table 4.** *Cont.*

**Table 4.** *Cont.*

Oligo-DNA microarrays were developed in common wheat, and these microarrays were designed to include approximately 32,000 distinctive genes characterized by several expressed sequence tags (ESTs). To classify the salinity-stress responsive genes, the expression profiles of transcripts that responded to stress were examined using microarrays. It was concluded that 5996 genes were verified by more than a 2-fold change in expression. These genes were categorized into twelve groups based on gene expression patterns [165]. Transcription-regulator activity, DNA binding, and the genes' assigned transcription factor functions were preferentially classified as immediate response genes. In wheat, candidate genes were identified as involved in salinity-stress tolerance [165,166]. These genes are active in the regulation of transcription [112,143] and the signal transduction that is engaged in metabolic pathways [167] or acting as ion transporters [168]. cDNA library in yeast (*Saccharomyces cerevisiae*) was examined using a synthetic medium augmented with excessive salt concentrations (900 mM). A few clones showed comparatively improved growth. The notorious clones bore the *Guanyl transferase* (OsMPG1) mannose-1-phosphate gene [133]. Extreme salinity stress was significantly linked with the transcription factors of four tomato genes from the family of *zinc finger*. There has been prior evidence of the relationship between *zinc finger* transcription factors and plant salinity tolerance [169,170]. Overexpression of *OSISAP1* in transgenic tobacco resulted in tolerance to salinity, dehydration, and cold stress in the new sprouts [171].

A microarray containing 384 genes associated with stress responses was used in *Medicago truncatula* genotypes (*Jemalong A17* and *108-R*) to compare rooting gene expression during salt stress. The homolog of flora *TFIIIA*-related TF, *MtZpt2*-1, and *COLD-REGULATEDA1* genes were known to regulate the previous genes and were acknowledged in *Jemalong A17* stress-tolerant genotypes. Two *MtZpt2* Transcription factors (*MtZpt2-1* and *MtZpt2-2*) have shown increased expression in the roots compared to *108-R* [172]. Salinity stress is attributed to diverse stresses that persuade overlapping patterns in gene expression. For example, in an investigation of 8100 *A. thaliana* genes, approximately 2400 genes were reported to have a widespread expression in exposure to salt, oxidative and cold stress [92]. In addition, 23 genes were reported against NaCl stress. This also accounted for a small percentage of DEGs, including encoding transcription factors *WOX2* and *BZIP3*, calcium-binding protein *CML42*, ubiquitin-protein ligase *UBC17*, and *IDA-like 5* protein [92]. Most prominently, synthesized *isiA* encoded a novel chlorophyll (*Chl*)-binding protein [173] (Table 3).


**Estitxu Villamor 1,2,\*, Ortzi Akizu-Gardoki 1,3, Olatz Azurza 1,4, Leire Urkidi 1,5, Alvaro Campos-Celador 6, Izaro Basurko 1,7 and Iñaki Barcena Hinojal 1,7**

Received: 22 January 2020; Accepted: 6 March 2020; Published: 12 March 2020

**Abstract:** Nowadays, there is a wide scientific consensus about the unsustainability of the current energy system and at the same time, social awareness about climate change and the IPCC's goals is increasing in Europe. Amongst the different pathways towards them, one alternative is the radical transition to a democratic low-carbon energy system where the local scale has a key leading role. Under this scope, this research is framed within the mPOWER project, financed by the European Commission's H2020 programme, which promotes collaboration among different European municipalities in order to boost the transition to a renewable-based participatory energy system. This paper presents the starting point of the mPOWER project, where the main energy features of 27 selected European municipalities are collected and analysed for the year 2016. An open public tender and selection process was carried out among European cities in order to choose the candidates to participate in mPOWER project. A view of this situation will be taken by the mPOWER project as a diagnostic baseline for the following steps: a peer-to-peer knowledge-sharing process among these European municipalities, and subsequently, among a more extensive group. The first finding of the paper is that, even if those municipalities are trying to reduce their greenhouse gas emissions, they are highly dependent on fossil fuels, even in cases where renewable energies have significant presence. Second, their energy consumption is logarithmically related to the human development index and gross domestic product but not to the size of the cities and their climate characteristics. Finally, despite the work that these cities are making towards energy transition in general and within the mPOWER project in particular, the paper shows a high difficulty mapping their energy systems. The lack of accurate and unified data by the municipalities is a sign of disempowerment at a local and public level in the energy sphere and makes difficult any strategy to advance towards a bottom-up energy transition. Among other goals, the mPOWER project aims to reveal these kinds of difficulties and help local authorities in managing their transition paths.

**Keywords:** energy transition; sustainable cities; transition roadmaps; renewable energies; policymaking; energy democracy; energy mapping

#### **1. Introduction**

The urgency for changing the current European energy model and transiting towards a more sustainable one is a well-accepted reality among European inhabitants, policy-makers and scientists. In line with this, it has been clearly detected that the elevated use of fossil fuels needs to be reduced in order to keep the temperature increase of the planet to under 2 degrees Celsius [1]. Furthermore, it has been accepted that the incoming transition will not be merely a technological transition towards renewable energy systems [2], but will require a change in our way of dealing with democracy, economy and social values [3,4]. Indeed, there is an on-going debate about how this multi-dimensional transition will take place [5]. The goals of energy democracy movements all over the world intend to resist the current energy agenda, and reclaim and restructure the energy sector [6], with desired outcomes such as shifting public resources away from fossil fuels, leaving fossil fuels on the ground and stopping extractivist infrastructures, ensuring public or social control of the energy sector, or not prioritising only the monetary benefit out of the energy system.

A number of different voices point to the need for a locally based energy transition, as the local scale is related to more participative, inclusive and socially accepted policies and actions. Van der Schoor et al. [7] argue that local communities should lead a bottom-up transition since they boost the use of local resources with democratic horizontal governance and own financial strength-based energy production and supply. Other similar studies maintain that the new energy system will be funded directly by citizens, since they are at the heart of this new transition based on decentralised renewable energy cooperatives [8]. In the same vein, Vita et el. [9] show that those individuals who are members of sustainability-oriented grassroots initiatives have a more sustainable lifestyle compared to their socio-demographic counterparts, which leads to a more satisfying life and lower carbon footprints in the analysed domains of housing, transport, clothing and food. Indeed, energy embedded in consumed products and services [10,11] is a major issue that goes beyond direct energy consumption and needs to be considered in local energy transitions in order to avoid global rebound effects. Beyond the influence of a single consumer [12], as Grabs et al. [13] point out, grassroots initiatives play a major role in this field. After analysing the nexus between individual motivation and collective action in the context of sustainable consumption, they concluded that individuals can be agents of societal change when they are organised in groups.

Furthermore, some authors attribute the progress of energy transitions in different countries to the presence of grassroots initiatives in such countries [14]. In the above-mentioned reference, Kooij et al. studied what the conditions are for grassroots initiatives to emerge, and how these initiatives create an impact upon these conditions. They observe that openness to alternative discourses and a shared knowledge are favourable conditions for the appearance of these kinds of initiatives. In turn, they argue that the influence they exert upon energy systems is low in the case of those systems with strong vested interests, and that the support by governments and institutions is crucial for those initiatives to make a change.

More specifically, in relation to local authorities, in the Sustainable Development Goals of 2015 [15], the United Nations clearly recognised the key role of local public institutions in the transition towards a more sustainable future. According to Sperling et al. [16], cities will be relevant in boosting locally produced and consumed energy systems based on renewables in different sectors. This change will occur by focusing our attention on underlying social drivers, and releasing the need for economic growth as a single scope [17]. However, they suggested that the role of municipalities needs to be outlined very clearly and that the state must provide municipalities with the necessary planning tools, establishing the required strategy, for the integration of a 100% renewable source-based decentralised

system [16] or decarbonisation plans [18]. Comodi et al. [19] show that, even while the role of local authorities is relevant, the results of their actions can be merely partial within a multi-scalar energy transition. The support and energy policies of nations and states remain central in achieving decarbonisation goals under democratic principles [20].

This paper has been developed within the mPOWER project, which focuses on the strategies and actions that municipal authorities carry out towards a sustainable and democratic energy system [21]. Within the framework of the project and despite their limitations, municipalities are recognised as key political actors in the transformation of the energy system within the European context.

During the last decade, the EU has promoted a proactive climate policy, increasing renewables and improving energy efficiency [22]. Some of the leading EU countries, going beyond climate change on the direction of the conservation of the national and global environment, have also opted for nuclear phasing-out [23]. However, the results do not appear to be sufficient. In Europe (European Union 28, EU-28), non-renewable energy consumption (coal, oil, gas and nuclear energy) constitutes 85.3% of the total primary energy supply [24], consuming 23.18 MWh of non-renewable energy per inhabitant and year (MWh·cap−1·yr−1) out of the total of 27.16 MWh·cap−1·yr−1. Even though the consumed non-renewable energy in EU-28 is 26% less than the average for OECD countries, it is 68.7% greater than the world average value [24], which has already been considered 2–6 times above sustainable levels, making it beyond the planetary boundaries [25]. This underlines that there is an urgency to continue reducing the consumed non-renewable energy in EU-28.

According to Tagliapietra et al. [22], the cost of a fully-fledged energy transition in the EU would be similar to that of preserving the current non-renewable energy system, and adopting the right policies to mitigate the distributional effects of said transformation, could make it also socially desirable. Since the social and metabolic transformations required by a real decarbonisation process are to be so relevant [26], it will be determinant to control its distributional and democratic aspects. Nevertheless, concerning local experiences, there is a gap in the current energy system between the new theoretical sustainable energy systems and the reality that the cities are facing [8]. In order to build bridges among theoretical roadmaps and practical strategies, the active role of the municipalities is considered a key factor [9]. Furthermore, difficulties in modelling the impacts of citizens' behaviour on climate change have been detected [27], concluding that further collaboration between social scientists and economic developers are required.

Cities are said to be responsible for 70% of global emissions of CO2eq [28], and different targets have been established in EU cities in order to face the energy transition towards a sustainable one. According to van den Dobbelsteen et al. [29], all targets at a municipal level should aim to research the current energy situation, reduce the total energy consumption, reuse energy (i.e., reuse flows, heat transfer) and produce renewable energy. To this effect, one of the first targets in the EU was established by the Swiss government, following the ETH researchers who estimate 17.5 MWh per capita per annum (equivalent of 2000 watt during 365 days and 24 h) as a sustainable amount [30]. This target is also aligned with the 1 eqtCO2 emissions per person and year, which would allow us to avoid a climate change scenario [30]. The percentage of integration of renewable energy or electricity has also been established, and most European cities have the goal of integrating 20% of renewable energy into their electric mix by 2020 [29]. The European Commission goes beyond and targets a renewable energy share of 20% in the gross final energy demand [31,32]. It has also been common to use relative targets in percentages, such as reductions of CO2eq emissions of 40% by 2030 [31,32] or 85–90% by 2050 [32] (compared to 1990). Another commonly used percentage target is a 27% increase in energy efficiency compared to a 'business-as-usual' projection of future energy demand [30,31]. The existence of different goals has generated a diversification of targets among different cities, with an absence of unified targets [33]. Nevertheless, despite the existence of targets, emission reductions in cities measured in consumption-based accounts do not always occur. In the study developed in six Japanese cities from 1980 to 2000, it was concluded that consumption-based emissions measured by carbon inventories have not been reduced, but rather increased from 8 to 9 eqtCO2 per year and capita [28]. Furthermore, it has been also detected that the energy consumption and carbon emissions of cities are related to their economic performance, especially in developed countries such as China [34], thus a massive reduction of energy or emissions could happen to not be attractive or convenient for some cities, drawing us into a controversial panorama.

Despite these limitations, different initiatives are being developed on the roadmap to more sustainable cities. One such example is the European Energy Awards [35], given to municipalities for energy and climate protection activities at the European level. Those cities awarded can be considered exemplary in energy transition.

In this regard, different bottom-up movements have arisen in recent years to boost the necessary energy transition. The 'Covenant of Mayors' (CoM) [36] is one of the most relevant initiatives, a movement that involves more than 9000 cities in 131 countries, where local authorities voluntarily commit to meeting and exceeding the European goals for CO2 emissions reductions [37]. In the same vein of work, 'Energy Cities', the European association of cities in energy transition, is a network of local authority representatives from 30 countries that gathers frontrunners and energy transition beginners, city officials and technical experts. Their principal goal is to support the creation of new policies through National Energy and Climate Plans (NECP) [38]. Similarly, Ursula von der Leyen presented the plan to make the EU the world's first 'climate-neutral' continent by 2050 [39].

Apart from the work that has been carried out on (and combined with) policy-making, from an academic perspective, several efforts to model a sustainable energy model have been made. The MEDEAS project [40] is one of the best examples, a tool to design the transition into a 100% renewable energy system (RES) in Europe. This initiative considers three different scenarios to model the energetic macro-economic system from 1995 to 2050: Business as Usual (BAU), Green Growth (GG) and the Post-Growth or degrowth approach (PG). Other tool to boost this transition in urban districts is the learning experience through the use of visualisation games, such as Go2Zero [41].

Within the framework of this European energy transition, the mPOWER project is funded by the European Commission Horizon 2020 Research and Innovation Framework Programme and aims to boost municipal actions, public engagement and the creation of routes towards the necessary Energy Transition [42]. The project is managed by seven partners (the University of Glasgow, Platform-London, the Stichting Transnational Institute, the Society for the Reduction of Carbon Limited, the Institute for Political Ecology, Energy Cities and the University of the Basque Country). Throughout the 48-month duration of the project, the partners will detect, through systematic peer review, the best replicable municipal practices in energy transitions and create a framework to share the different achievements in a peer-to-peer learning programme.

The central question of this paper is: What is the current situation, regarding energy, of the 27 municipalities that were selected for the initial stages of the learning programme? In addition, two sub-questions are defined, to be answered throughout the structure of the paper:

The analysis used in order to answer these questions is based on the statistical evaluation of various energy indicators and was carried out using the data provided by municipalities from an online survey related to energy consumption, renewable production and municipal policies and strategies of participatory energy transition. The data were collected in order to determine the baseline for year 2016 (in some cases, updated data of 2017 have been used), that is, the reference situation that will be compared with that of the end of the project. To this end, first, a description is given of the mPOWER project, as well as the methodology used in the research, which includes the baselining, standardisation and evaluation of the data. Following this, the results obtained from the 27 cities (i.e., municipalities) are presented and, finally, a summary provided of the conclusions drawn.


**Joseph J. Noh, Myeong-Seon Kim, Young-Jae Cho, Soo-Young Jeong, Yoo-Young Lee and Ji-Yoon Ryu et al.**

Anti-Cancer Activity of As4O6 and its Efficacy in a Series of Patient-Derived Xenografts for Human Cervical Cancer

Reprinted from: *Pharmaceutics* **2020**, *12*, 987, doi:10.3390/pharmaceutics12100987 . . . . . . . . . **323**


**Maria Angela Perito 1,2,\*, Silvia Coderoni 3 and Carlo Russo 4**

Received: 4 September 2020; Accepted: 16 September 2020; Published: 20 September 2020

**Abstract:** Food made with upcycled ingredients has received considerable attention in very recent years as a result of the need to both reduce waste and increase food nutritional properties. However, consumer acceptance of these novel foods is fundamental to their market uptake. This paper aims to assess the likelihood of the acceptance of food obtained from upcycled ingredients of olive oil productions and its association with some relevant recent consumption trends, such as organic food consumption and attention to food origin. In addition, particular attention is given to age group behaviors to appraise the differences between generations. Results suggest that, despite the negative influence of food technophobia, a core of sustainability-minded consumers seems to emerge that is interested in organic or local products, that could also favor the uptake of these novel food made with upcycled ingredients in the market. Results suggest that developing organic or "local" food products with upcycled ingredients can increase the probability of consumer acceptance.

**Keywords:** olive leaves; organic; local; consumer attitude; up-cycled ingredients; by-products; generational differences


**Acknowledgments**

We would like to thank Wei-Ju Chen, Xun Luo and Wenze Zhang for reviewing and abstracting data from Chinese publications. Financial support for this review was provided by the Bill and Melinda Gates Foundation to the US Fund for UNICEF for the ongoing work of the Child Health Epidemiology Reference Group (CHERG).


**Absorption and Transport of Inorganic Carbon in Kelps with Emphasis on** *Saccharina japonica*

Yanhui Bi and Zhigang Zhou

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62297

#### **Abstract**

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of Botany. 2002;89:887–894. DOI: 10.1093/aob/mcf064

S0003701X12010161

110 Applied Photosynthesis - New Progress

Due to the low CO2 concentration in seawater, macroalgae including *Saccharina japonica* have developed mechanisms for using the abundant external pool of HCO3 − as an exogenous inorganic carbon (Ci ) source. Otherwise, the high photosynthetic efficiency of some macroalgae indicates that they might possess CO2 concentrating mechanisms (CCMs) to elevate CO2 concentration intracellularly around the active site of ribu‐ lose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCo). As the photosynthetic modes of macroalgae are diverse (C3, C4 or a combination of C3 and C4 pathway), CCMs in different carbon fixation pathways should vary correspondingly. However, both in C3 and C4 pathways, carbonic anhydrase (CA) plays a key role by supplying either CO2 to RuBisCO or HCO3 − to PEPC. Over the past decade, although CA activities have been detected in a number of macroalgae, genes of CA family, expression levels of CA genes under different CO2 concentrations, as well as subcellular location of each CA have been rarely reported. Based on analysis the reported high-throughput sequencing data of *S. japonica*, 12 CAs of *S. japonica* (*SjCA*) genes were obtained. Neighbor-Joining (NJ) phylogenetic tree of SjCAs constructed using Mega6.0 and the subcellular location prediction of each CA by WoLFPSORT are also conducted in this article.

**Keywords:** Macroalgae, Inorganic carbon uptake, C3 and C4 metabolism, Carbonic anhydrase, *Saccharina japonica*

### **1. Introduction**

Kelps demonstrate high photosynthetic rates. According to the reports, productivity of large brown algae (e.g., *Macrocystis*, *Laminaria*, *Ecklonia*, *Sargassum*) ranges from 1000 to 3400 g m−2yr −1C or about 3300 to 11,300 g m−2yr−l dry weight, and red algae show a similar range of produc‐

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

tion. Cultivated macroalgae can yield even higher values. The projected yield of cultivated *Laminaria japonica* on an annualized basis is equivalent to 1300 t ha−1 fresh weight or 6.5 times the maximum projected yield for sugarcane, the most productive of land plants under cultiva‐ tion. In general, 45% yield of the dry weight of plants is accounted by carbon, which is assimi‐ lated in plant through Calvin cycle. The high productivities of kelps indicate their higher photosynthetic efficiency than C4 terrestrial plants [1].

The enzyme ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCo) is crucial in CO2 assimilation. This bifunctional enzyme could catalyse the initial steps of photosynthetic carbon reduction and photorespiratory carbon oxidation cycles by combining CO2 and O2 with ribulose-1, 5-bisphosphate (RuBP) [2, 3]. RuBP carboxylation determines the net photosyn‐ thetic efficiency of photoautotrophs [4]. However, RuBisCo has a surprisingly low affinity for CO2 and the oxygenase activity is intrinsic to RuBisCo. For kelps, the enzymatic efficiency of RuBisCo is also limited by the low concentration and diffusion coefficient of CO2 in seawater [5]. At a natural pH of about 8, the major part of the dissolved inorganic carbon (DIC) is in the form of bicarbonate (HCO3 − ), and only about 12 μM is present as dissolved CO2 [6], which is much lower than the half-saturation constant (Ks) of RuBisCo for CO2 ranges from 30 μM to 60 μM in marine macroalgae [7, 8]. To support photosynthesis and growth, seaweeds require an exogenous inorganic carbon (Ci ), while only CO2 and HCO3 − can be used as a CO2 source for photosynthesis. Due to the low CO2 concentration in seawater, it is not surprising that most seaweed have developed mechanisms for using the abundant external pool of HCO3 − as an exogenous Ci source [9–11]. And it seems likely that those macrophytes that are able to use HCO3 − would possess advantages compared with that rely solely on diffusive CO2 entry. Here the question is how Ci is absorbed, transported to supply high CO2 concentration around RuBisCo in kelps since unlike CO2, HCO3 − cannot diffuse through the lipid bilayer of the plasma membrane [12] and the produced or absorbed CO2 are readily leaked out due to the high CO2 permeability of cytomembrane. Otherwise, different models of photosynthesis such as C3, C4 and CAM might employ different CCMs in kelps. Thus, this review mainly focuses on the mechanisms of Ci absorption, transportation and concentration mechanisms of multicel‐ lular marine algae, including representatives of Chlorophyceae, Rhodophyceae and Phaeo‐ phyceae with different photosynthetic types*.*

#### **2. Photosynthetic modes of macroalgae**

As with terrestrial angiosperms where a single family may possess species with divergent photosynthetic modes [13], the marine macroalgal divisions also exhibit diversity. The photosynthetic carbon fixation pathways of marine macrophytic algae generally follow that of C3 plants [14]. However, for certain genera, a number of studies have shown photosynthesis to possess C4-like photosynthetic characteristics, including the high phosphoenolpyruvate carboxykinase (PEPCK) activity with low phosphoenolpyruvate carboxylase (PEPC) activity, little photorespiration and the labelling of malate and aspartate as an early product of carbon fixation. Based on this, it has been suggested that these macroalgae are of the C4 type, or a combination of C3 and C4, type [15–17], although Kremer and Küppers [18] had contradicted the decision whether a species is a C4 plant or not based only on chromatographic and enzymatic analysis. In recent decades, our understanding of the possible metabolic pathways of macroalgae has been extended with using the available sequencing resources and molecular technologies and applying molecular approaches. Reiskind et al. [19] reported that a limited C4-like system in the green alga *Udotea* with the high PEPCK activity and low PEPC activity was a novel characteristic. Whereafter, Reiskind and Bowes [20] found that when PEPCK activity was inhibited *in vivo* with 3-mercaptopicolinic acid, thallus photosynthesis was decreased by 70% and the labelling of early photosynthetic products such as malate and aspartate was reduced by 66% and thus provided new evidences for the existence of C4 acid metabolism in this green alga. In contrast to *Udotea*, *Codium*, a macroalga closely related to *Udotea*, exhibits gas exchange characteristics that resemble terrestrial C3 plants, and neither C4 acids nor PEPCK plays a part in photosynthesis [19]. This demonstrates the diversity of photosynthetic mechanisms in the Chlorophyta. *Ulva*, a common green seaweed, was previ‐ ously reported as a typical C3 plant based on some biochemical evidences that 3-phosphogly‐ ceric acid (3-PGA) was the main primary product formed photosynthetically and a high RuBPcase/PEPcase ratio was found in it [21], while, recently, it was reported that *Ulva* possessed rather comprehensive carbon fixation pathways including C3, C4 and CAM mechanisms because key genes of enzymes involved in these photosynthetic modes were got from the expressed sequence tag (EST) using Kyoto encyclopedia of genes and genomes (KEGG) [22]. Recently, C4-like carbon fixation pathway was also found in representatives of Rhodophyceae and Phaeophyceae based on the analysis of ESTs or transcriptomes. In red algae, Fan et al. [23] speculated that the sporophyte of *Pyropia haitanensis* most likely possesses a C4-like carbon fixation pathway since genes of the key enzymes in the PCK-type C4 carbonfixation pathway were abundantly transcribed*.* Wang et al. [24] assumed that a C4-like carbonfixation pathway might play a special role in fixing inorganic CO2 in *Porphyra yezoensis* with the evidence that except pyruvate-phosphate dikinase all genes involved in C4-pathway were discovered from the transcriptome. Xu et al. [25] had reported that PEPCK, an important enzyme in carbon fixation in C4 plants, had very high activity in the sporophyte of *L. japoni‐ ca*. Besides, haploid gametophytes and diploid sporophytes of some marine macroalgae with dimorphic life cycles might even employ different photosynthetic mode. Wang et al. [24] found that both the RuBisCo content and the initial carboxylase activity were notably higher in gametophytes than in the sporophytes of four seaweed species — *P. yezoensis*, *P. haitanensis*, *Bangia fuscopurpurea* (Rhodophyte) and *L. japonica* (Phaeophyceae). They assumed that in the sporophyte of these algae, the major carbon fixation pathway may be a C4-like carbon fixation pathway, and thus a high abundance of RuBisCo would not be necessary for the sporophytes. And for *L. japonica*, the higher RuBisCo content and activity in gametophyte was corresponding to the lower photosynthetic rate, which implied there might be a greater difference between sporophytes and gametophytes of this alga in their photosynthetic mode. Conclusively, the existence of C4-like pathway in macroalgae has been verified using more evidence, while the distribution between C3 and C4 pathways was unknown during growth of macroalgae with comprehensive carbon fixation pathways including C3 and C4.

tion. Cultivated macroalgae can yield even higher values. The projected yield of cultivated *Laminaria japonica* on an annualized basis is equivalent to 1300 t ha−1 fresh weight or 6.5 times the maximum projected yield for sugarcane, the most productive of land plants under cultiva‐ tion. In general, 45% yield of the dry weight of plants is accounted by carbon, which is assimi‐ lated in plant through Calvin cycle. The high productivities of kelps indicate their higher

The enzyme ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCo) is crucial in CO2 assimilation. This bifunctional enzyme could catalyse the initial steps of photosynthetic carbon reduction and photorespiratory carbon oxidation cycles by combining CO2 and O2 with ribulose-1, 5-bisphosphate (RuBP) [2, 3]. RuBP carboxylation determines the net photosyn‐ thetic efficiency of photoautotrophs [4]. However, RuBisCo has a surprisingly low affinity for CO2 and the oxygenase activity is intrinsic to RuBisCo. For kelps, the enzymatic efficiency of RuBisCo is also limited by the low concentration and diffusion coefficient of CO2 in seawater [5]. At a natural pH of about 8, the major part of the dissolved inorganic carbon (DIC) is in the

much lower than the half-saturation constant (Ks) of RuBisCo for CO2 ranges from 30 μM to 60 μM in marine macroalgae [7, 8]. To support photosynthesis and growth, seaweeds require

photosynthesis. Due to the low CO2 concentration in seawater, it is not surprising that most seaweed have developed mechanisms for using the abundant external pool of HCO3

membrane [12] and the produced or absorbed CO2 are readily leaked out due to the high CO2 permeability of cytomembrane. Otherwise, different models of photosynthesis such as C3, C4 and CAM might employ different CCMs in kelps. Thus, this review mainly focuses on

lular marine algae, including representatives of Chlorophyceae, Rhodophyceae and Phaeo‐

As with terrestrial angiosperms where a single family may possess species with divergent photosynthetic modes [13], the marine macroalgal divisions also exhibit diversity. The photosynthetic carbon fixation pathways of marine macrophytic algae generally follow that of C3 plants [14]. However, for certain genera, a number of studies have shown photosynthesis to possess C4-like photosynthetic characteristics, including the high phosphoenolpyruvate carboxykinase (PEPCK) activity with low phosphoenolpyruvate carboxylase (PEPC) activity, little photorespiration and the labelling of malate and aspartate as an early product of carbon

−

), while only CO2 and HCO3

− would possess advantages compared with that rely solely on diffusive CO2 entry. Here

source [9–11]. And it seems likely that those macrophytes that are able to use

is absorbed, transported to supply high CO2 concentration around

absorption, transportation and concentration mechanisms of multicel‐

cannot diffuse through the lipid bilayer of the plasma

), and only about 12 μM is present as dissolved CO2 [6], which is

−

can be used as a CO2 source for

− as an

photosynthetic efficiency than C4 terrestrial plants [1].

−

form of bicarbonate (HCO3

112 Applied Photosynthesis - New Progress

exogenous Ci

the question is how Ci

the mechanisms of Ci

HCO3

an exogenous inorganic carbon (Ci

RuBisCo in kelps since unlike CO2, HCO3

phyceae with different photosynthetic types*.*

**2. Photosynthetic modes of macroalgae**

In C3 and C4 metabolisms, CO2 is the substrate of RuBisCo and assimilated through the Calvin cycle. In this cycle, CO2, catalysed with RuBisCo, combines with RuBP to form two molecules of 3-PGA. PGA is reduced to triose. RuBisCo, a bifunctional enzyme, may catalyse the combination of RuBP and CO2 for photosynthetic carbon reduction or may combine with O2 for C2 photorespiration [3]. The ratio of CO2 to O2 around RuBisCo is a major factor for the enzyme to choose the photosynthetic carbon reduction or C2 photorespiration carbon oxidation [26]. The low CO2 concentration around RuBisCo may not only impose restrictions on photosynthesis but also cause permanent light injuries to photosynthetic organelle [27–29]. The speciation of DIC (Ci ) is pH dependent. Above pH 4.5, the proportion occurring as CO2 (aq) decreases and HCO3 − increases, while above pH 8.3, the bicarbonate equivalence point, the equilibrium begins to shift towards carbonate (CO3 2− ). In the upper layer of the oceans, HCO3 − ions predominate, and the dissolved CO2 represents only about 1% of the total dissolved carbon with a concentration of about 21 μM [30]. The *K*m (CO2) value of RuBisCO is signifi‐ cantly higher than this, having been reported as being as high as 200 μM in some cyanobacteria [31]. To survive under the selective pressure of low CO2 concentration, high permeability of CO2 for plasma membrane and low affinity of CO2 for RuBisCo, many algae, including macroalgae living in the subtidal zone, have evolved with inorganic CCM that allows them to overcome this potentially limiting shortage of CO2 [9, 32–36]. So, the productivity of most macroalgae is not currently considered limited by DIC. Unlike terrestrial C4 plants possessing Kranz anatomy to prevent futile recycling of CO2 by segregating the initial carboxylation and decarboxylation reactions in different cells, macroalgae concentrate CO2 internally, which is mediated by Ci transporters at the plasma membrane or chloroplast envelope and CA. As for carboxylases are different between C3 and C4 metabolism, Ci acquisition, transportation and concentration mechanisms might be diverse.

Based on a series of reports on the presence of CCM in blue-green algae and *Chlamydomonas* (*Chlamydomonas reinhardtii*) and some other microalgae [37–40], Badger [41] reported that the CCM of algae possess at least three functional elements: (1) the transportation of the Ci dissolved in seawater into cells in the form of CO2 and/or HCO3 − ; (2) the accumulation of the Ci in cells in the form of HCO3 − , forming pools of the dissolved Ci and (3) the delivery of CO2 to the periphery of RuBisCo from such pools.

#### **3. Inorganic carbon absorption mechanisms of macroalgae**

The methods of CO2 and/or HCO3 − absorption of macroalgae cells (Figure 1) include the following: (1) non-CCM macroalgae (that do not possess or use CCM) rely exclusively on diffusive uptake of CO2, (2) CCM macroalgae uptake of Ci , as CO2 and/or HCO3 − via mechanisms of the external carbonic anhydrase (CAext) mechanism, the anion exchange (AE) transport mechanism, the plasma membrane associated with H+ -ATPase mechanism and passive transport of CO2 by diffusion. In the first mechanism, HCO3 − in the periplasmic space is converted to CO2 at the presence of CAext, an enzyme that is located in the cell wall in the

In C3 and C4 metabolisms, CO2 is the substrate of RuBisCo and assimilated through the Calvin cycle. In this cycle, CO2, catalysed with RuBisCo, combines with RuBP to form two molecules of 3-PGA. PGA is reduced to triose. RuBisCo, a bifunctional enzyme, may catalyse the combination of RuBP and CO2 for photosynthetic carbon reduction or may combine with O2 for C2 photorespiration [3]. The ratio of CO2 to O2 around RuBisCo is a major factor for the enzyme to choose the photosynthetic carbon reduction or C2 photorespiration carbon oxidation [26]. The low CO2 concentration around RuBisCo may not only impose restrictions on photosynthesis but also cause permanent light injuries to photosynthetic organelle [27–29].

) is pH dependent. Above pH 4.5, the proportion occurring as CO2

increases, while above pH 8.3, the bicarbonate equivalence point, the

). In the upper layer of the oceans, HCO3

acquisition, transportation and

; (2) the accumulation of the

−

in the periplasmic space is

via mechanisms

−

absorption of macroalgae cells (Figure 1) include the

−

, as CO2 and/or HCO3

, forming pools of the dissolved Ci and (3) the delivery of CO2

−

2−

ions predominate, and the dissolved CO2 represents only about 1% of the total dissolved carbon with a concentration of about 21 μM [30]. The *K*m (CO2) value of RuBisCO is signifi‐ cantly higher than this, having been reported as being as high as 200 μM in some cyanobacteria [31]. To survive under the selective pressure of low CO2 concentration, high permeability of CO2 for plasma membrane and low affinity of CO2 for RuBisCo, many algae, including macroalgae living in the subtidal zone, have evolved with inorganic CCM that allows them to overcome this potentially limiting shortage of CO2 [9, 32–36]. So, the productivity of most macroalgae is not currently considered limited by DIC. Unlike terrestrial C4 plants possessing Kranz anatomy to prevent futile recycling of CO2 by segregating the initial carboxylation and decarboxylation reactions in different cells, macroalgae concentrate CO2 internally, which is mediated by Ci transporters at the plasma membrane or chloroplast envelope and CA. As for

Based on a series of reports on the presence of CCM in blue-green algae and *Chlamydomonas* (*Chlamydomonas reinhardtii*) and some other microalgae [37–40], Badger [41] reported that the CCM of algae possess at least three functional elements: (1) the transportation of the Ci

following: (1) non-CCM macroalgae (that do not possess or use CCM) rely exclusively on

of the external carbonic anhydrase (CAext) mechanism, the anion exchange (AE) transport

converted to CO2 at the presence of CAext, an enzyme that is located in the cell wall in the

The speciation of DIC (Ci

114 Applied Photosynthesis - New Progress

−

equilibrium begins to shift towards carbonate (CO3

carboxylases are different between C3 and C4 metabolism, Ci

dissolved in seawater into cells in the form of CO2 and/or HCO3

**3. Inorganic carbon absorption mechanisms of macroalgae**

−

−

diffusive uptake of CO2, (2) CCM macroalgae uptake of Ci

mechanism, the plasma membrane associated with H+

transport of CO2 by diffusion. In the first mechanism, HCO3

concentration mechanisms might be diverse.

to the periphery of RuBisCo from such pools.

Ci in cells in the form of HCO3

The methods of CO2 and/or HCO3

(aq) decreases and HCO3

**Figure 1.** A schematic diagram on the photosynthetic carbon physiology of some macroalgae revised from [45].

majority of seaweeds and could be inhibited by the membrane impermeable acetazolamide (AZ), and then the resulting CO2 is readily taken into the cell by passive diffusion. This seems to be the most prevalent for HCO3 − utilization among seaweeds [42, 43], but it may be nonfunctional under high pH (>9.00) [44, 45]. The AE transport mechanism is HCO3 − direct uptake through the AE protein in plasma membrane [11, 43, 46–48], which is 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) sensitive. This operates equally well at pH 8.4 and 9.4 [44, 45]. H+ -ATPase mechanism refers to a plasma membrane associated H+ -ATPase pump that extrudes the excess cellular H+ to the outside of the plasma membrane facilitating a H+ / HCO3 − co-transportation or enhancement of the external uncatalysed dehydration of HCO3 − to CO2 in the periplasmic space [49]. However, this has only been reported in some Laminariales such as *S. latissima* and *L. digitata*. Along with the uptake of CO2 and/or HCO3 − , the internal charge balance (OH− /H+ ) will be absolutely changed. To maintain intracellular ion balance, macroal‐ gae employ diverse strategies. In AE mechanism, the active transport of HCO3 − into the cell might result in an outward flux of OH− [50–53, 45] as this mechanism is involved in a one-forone exchange of anions across the plasma membrane. The OH− efflux can increase H+ in the cell [52]. To maintain the intracellular OH− /H+ balance, H+ extrusion might be required. In macroalgae possessing H+ -ATPase mechanism, their plasma membrane associated with H+ - ATPase pump might extrude excess cellular H+ to the outside of the plasma membrane, while in macroalgae that do not have H+ -ATPase pump in their plasma membrane, the regulation of intracellular ion balance might be related to a high activity of internal carbonic anhydrase (CAint), including the CA in cytoplasm, chloroplast stroma, thylakoid lumen and mitochondria [45].

The extent to which marine macroalgae are able to acquire HCO3 − for photosynthesis varies among taxa and/or species, and the special strategies by which the alga acquire Ci is closely related to habitat including pH and depth, conferring as adaptation advantage to the alga [9, 33, 36, 54–56]. Cornwall et al. [57] reported when light is low, CCM activity of macroalgae is reduced in favour of diffusive CO2 uptake and the proportion of non-CCM (diffusive uptake of CO2) species increased with depth. Otherwise, pH might also control Ci use by macroalgae. In *U. lactuca*, the CAext-mediated mechanism is the main method of HCO3 − utilization under normal pH conditions, whereas when they were grown at high pH, direct uptake of HCO3 − via a DIDS-sensitive mechanism can be induced [44]. Similar HCO3 − utilizing mechanisms were found in another green macroalgae *Enteromorpha intestinalis* [54]. For the red alga *Gracilaria gaditana*, the HCO3 − use is also carried out by the two DIC uptake mechanisms, in which the indirect use of HCO3 − by an external CA activity being the main pathway and the potential contribution to HCO3 − acquisition by the DIDS-sensitive AE mechanism was higher after culturing at a high pH [58]. However, these two mechanisms do not occur simultaneously, and the DIDS-sensitive mechanism is induced only under high pH. *Solieria filiformis*, another red marine macroalgae, in which the general form of Ci transported across the plasma membrane is CO2, but HCO3 − acquisition takes place simultaneously between CAext mechanism and direct uptake [59]. CAext mechanism is also the main pathway for DIC acquisition for the species of Phaeophyta. *S. latissima* mainly uses CAext mechanism for HCO3 − absorption, since when AZ is used to treat *S. latissima*, its photosynthetic efficiency drops by 80% [11]. Otherwise, *S. latissima* also has a H+ -ATPase mechanism, of which the proton pump may support the antiport of H+ / HCO3 − or the discharge of H+ , creating an acid environment in the periplasmic space and causing the dehydration of HCO3 − into CO2 with CA to quickly diffuse into cells [49]. *S*imilar to *S. latissima*, *L. digitata* also has a CAext mechanism of absorbing HCO3 − and a P-H+ - ATPase mechanism [49]. Gametophytes of *Ectocarpus siliculosus* utilize the CAext mechanism and the HCO3 − transport protein [60] on the cell membrane to absorb HCO3 − . *Macrocystis pyrifera* utilizes the CAext mechanism and the AE protein mechanism to absorb HCO3 − , in which the main mechanism of HCO3 − uptake is via AE protein and CAext contributes little [45]. For *Sargassum henslowianum*, like most seaweed, the main Ci acquisition strategy is also CAext metabolism, since its photosynthetic O2 evolution could be drastically depressed by AZ at pH 8.1 (i.e., the normal seawater pH value) and at pH 9.0. And direct uptake for HCO3 − via DIDSsensitive AE protein mechanism was unlikely to be present in Ci acquisition of this kelp, because the photosynthesis in either blade or receptacle tissue of this alga was not affected by DIDS [61]. For *Hizikia fusiformis*, CAext+ diffusive uptake of CO2 could support its metabolic requirements sufficiently since there is no known other active Ci transport mechanisms [62]. For *S. japonica*, Yue et al. [63] found that the Ci absorption of the CAext mechanism in its juvenile sporophytes accounts for 75% of the total Ci absorption in algae cells, whereas free CO2 absorption accounts for 25% only.

Thus, the CAext mechanism plays an important role in the CCM macroalgae absorption and the utilization of the relatively abundant HCO3 − in seawater.

#### **4. Ci transition process in CCMs of macroalgae**

ATPase pump might extrude excess cellular H+ to the outside of the plasma membrane, while

intracellular ion balance might be related to a high activity of internal carbonic anhydrase (CAint), including the CA in cytoplasm, chloroplast stroma, thylakoid lumen and mitochondria

related to habitat including pH and depth, conferring as adaptation advantage to the alga [9, 33, 36, 54–56]. Cornwall et al. [57] reported when light is low, CCM activity of macroalgae is reduced in favour of diffusive CO2 uptake and the proportion of non-CCM (diffusive uptake of CO2) species increased with depth. Otherwise, pH might also control Ci use by macroalgae.

normal pH conditions, whereas when they were grown at high pH, direct uptake of HCO3

found in another green macroalgae *Enteromorpha intestinalis* [54]. For the red alga *Gracilaria*

culturing at a high pH [58]. However, these two mechanisms do not occur simultaneously, and the DIDS-sensitive mechanism is induced only under high pH. *Solieria filiformis*, another

and direct uptake [59]. CAext mechanism is also the main pathway for DIC acquisition for the

when AZ is used to treat *S. latissima*, its photosynthetic efficiency drops by 80% [11]. Otherwise,

ATPase mechanism [49]. Gametophytes of *Ectocarpus siliculosus* utilize the CAext mechanism

*Sargassum henslowianum*, like most seaweed, the main Ci acquisition strategy is also CAext metabolism, since its photosynthetic O2 evolution could be drastically depressed by AZ at pH

because the photosynthesis in either blade or receptacle tissue of this alga was not affected by DIDS [61]. For *Hizikia fusiformis*, CAext+ diffusive uptake of CO2 could support its metabolic

transport protein [60] on the cell membrane to absorb HCO3

−

*S*imilar to *S. latissima*, *L. digitata* also has a CAext mechanism of absorbing HCO3

*pyrifera* utilizes the CAext mechanism and the AE protein mechanism to absorb HCO3

8.1 (i.e., the normal seawater pH value) and at pH 9.0. And direct uptake for HCO3

use is also carried out by the two DIC uptake mechanisms, in which the

by an external CA activity being the main pathway and the potential

acquisition by the DIDS-sensitive AE mechanism was higher after

acquisition takes place simultaneously between CAext mechanism

uptake is via AE protein and CAext contributes little [45]. For

among taxa and/or species, and the special strategies by which the alga acquire Ci

In *U. lactuca*, the CAext-mediated mechanism is the main method of HCO3

species of Phaeophyta. *S. latissima* mainly uses CAext mechanism for HCO3

The extent to which marine macroalgae are able to acquire HCO3

a DIDS-sensitive mechanism can be induced [44]. Similar HCO3

red marine macroalgae, in which the general form of Ci

− or the discharge of H+

−

sensitive AE protein mechanism was unlikely to be present in Ci

−

− for photosynthesis varies

−

− utilizing mechanisms were

transported across the plasma

−

, creating an acid environment in the periplasmic

into CO2 with CA to quickly diffuse into cells [49].

is closely

− via

utilization under

absorption, since

and a P-H+

. *Macrocystis*

, in which

via DIDS-

−

−

−

−

acquisition of this kelp,

in macroalgae that do not have H+

116 Applied Photosynthesis - New Progress

[45].

*gaditana*, the HCO3

indirect use of HCO3

contribution to HCO3

membrane is CO2, but HCO3

*S. latissima* also has a H+

−

the main mechanism of HCO3

antiport of H+ / HCO3

and the HCO3

−

−

−

space and causing the dehydration of HCO3

Ci acquisition mechanisms are extensively studied and well-known in microalgae [44, 38]. For instance, regardless of the Ci form (CO2 or HCO3 − ) taken up by the microalga *C. reinhardtii*, HCO3 − is the primary form accumulated into the cell to prevent CO2 leakage [38]. In macroalgae, most Ci use processes are speculated based on some biochemical evidence. For C3 photosynthesis, the CO2 that entered the cytoplasm is transformed into HCO3 − under the catalytic action of CA in the cytoplasm and stored in the cytoplasm [38] to maintain the equilibrium of different forms of Ci and to regulate the pH value of the cytoplasm [26, 38]. The HCO3 − in the cytoplasm enters the chloroplast stroma via the Ci transport protein on the chloroplast membrane, and the CO2 in the cytoplasm directly enters the stroma via the chloroplast membrane. In diatom *Phaeodactylum tricornutum*, genes with homology to bicarbonate transporters from SLC4 and SLC6 families, two HCO3 − transporters studied thoroughly in human, were got from its genome and one of these SLC4-type HCO3 − transporters has recently been confirmed to function as a Na+ -dependent HCO3 − transporter on the outer membrane [64, 65]. However, the molecular nature of HCO3 − transporters of macroalgae is unknown now, and their similarity to those found in diatoms is uncertain. The transportation of Ci from the cytoplasm to the chloroplast is the major Ci flux in the cell and the primary driving force for the CCM. This flux drives the accumulation of Ci in the chloroplast stroma and generates a CO2 deficit in the cytoplasm, inducing CO2 influx into the cell. Given that the pH value of the chloroplast stroma is closer to 8, the stroma Ci is mostly enriched in the form of HCO3 − , forming Ci pools [66]. In macroalgae, which have pyrenoids, HCO3 − is putatively carried into the thylakoid by the Ci transport protein on the thylakoid membrane, forming CO2 in the thylakoid space under the catalytic action of thylakoid CA [67, 68]. The thylakoid membrane partially sinks into the pyrenoids [69], where the diffused CO2 is quickly fixed by the RuBisCo in the pyrenoids. The diffused CO2 from the thylakoid space outside the pyrenoids or the unfixed CO2 leaked from the pyrenoids is transformed into HCO3 − under the action of CA in the starch sheath on the periphery of the pyrenoid, thus increasing the number of HCO3 − pools in the matrix [70]. For macroalgae without pyrenoids, such as *L. japonica*, HCO3 − entered the chloroplast stroma after being dehydrated under the action of chloroplast stroma CA and provided CO2 for the RuBisCo in the matrix (Figure 1).

For C4 photosynthesis, CA is required to convert CO2 to HCO3 − in the cytosol, and thus supply PEPC with substrate. HCO3 − will be fixed into malate. For non-PEPC algae with PEPCK, the CO2 entering the cytoplasm will be directly fixed in the form of four-carbon acid [71]. The produced four-carbon acid may be transported into the mitochondria, forming pyruvate after decarboxylation and CO2 release, which is fixed in the form of carbohydrate in the Calvin cycle. In fact, the presence of CA in C4 plants has been suggested to accelerate the rate of photosyn‐ thesis in C4 plants 104 -fold over what it would be if this enzyme were absent [72].

In conclusion, CA (CAext+CAint) is essential for the reversible HCO3 − –CO2 conversion both in the cell and in the periplasm. They participate in photosynthesis by supplying either CO2 to RuBisCO or HCO3 − to PEPC for C4 type.


*3.4. Concentration, Uptake, and Translocation of Vanadium*

This study depicted that V treatment significantly raised the V concentration in both shoots and roots, and maximum concentrations were recorded in 75 mg L−1 treated plants; 32.03 mg kg−1 DW in the shoots of sweet potato and 52.68 mg kg−1 DW in the roots of sweet potato (Table 3). Similarly, the accumulation of V was found to be higher in the roots than in the shoots. Moreover, V uptake by shoots and roots of sweet potato significantly augmented as the level of V increased. In the same way, the translocation of V from root to shoot was also raised significantly by the increment of V concentration (Table 3).

**Figure 2.** The influence of various V treatments on root morphological traits; (**A**) root length, (**B**) root volume, (**C**) surface area, (**D**) average diameter, (**E**) projected area, (**F**) number of root tips per plant, (**G**) number of root forks per plant, (**H**) number of root crossings per plant, and (**I**) root length per volume. Duncan's test specifies a significant difference (*p* < 0.05) between the means of the five treatments indicated by different letters.

**Figure 3.** The influence of various V treatments on leaf gas exchange elements; (**A**) transpiration rate, (**B**) photosynthetic rate, (**C**) stomatal conductance, and (**D**) intercellular CO2. Duncan's test indicates a significant difference (*p* < 0.05) between the means of the five treatments indicated by different alphabets.

Duncan's test indicates a significant difference (*p* < 0.05) between the means of the five treatments indicated by different alphabets.

### *3.5. Photosynthetic Pigments*

The chlorophyll (Chl) was significantly influenced by the application of V stress. Compared to the control, adding 10 mg L−1 V did not significantly raise the concentration of chlorophyll and carotenoid (Car) (Figure 4). On the other hand, higher treatment of V (25, 50, and 75 mg L−1) showed a significant negative impact on the photosynthetic pigments, and utmost reduction was detected at 75 mg L−1. Compared to the control, 77% reduction in the content of total chl, 65.9% in chl a, 70.5% in chl b, and 50.3% in Car were detected (Figure 4).

**Figure 4.** Influence of various V treatments on the photosynthetic pigments in the leaves of sweet potato. (**A**) Total chlorophyll content (T. Chl), (**B**) chlorophyll a (chl a), (**C**) chlorophyll b (chl b), and (**D**) carotenoid (Car) content. Duncan's test indicates a significant difference (*p* < 0.05) between the means of the five treatments indicated by different alphabets.


**2. Art and medicine**

As a result of reading history, we can be aware of how much art and medicine have remained integrated for centuries. It is enough to go far back in time to find examples transmitted by artistic objects to understand, as a whole, the importance of the solid relationship between medicine and visual arts starting from when in ancient Greece, which can be considered the cradle of our modern culture, the anatomists asked the artists for help to understand the human body. In fact, at that time, dissection was practiced on the bodies of animals, while it was forbidden to explore human anatomy for social and religious reasons.

Respect for the body of the deceased on the one hand and the consideration of the corpse as a source of impurity on the other had meant that the conditions were not created for carrying out this type of investigation. Even after 1241, the year of the edict of Frederick II which authorized, and indeed stimulated, the use of dissection of corpses, the anatomical investigation was accompanied throughout its path by the activity of artists who have put their skills at the service of scientific studies and of the representation of the human body, which needed scientific investigation to be able to fully express itself.

The practice of observation and the in-depth study of shapes allowed them to reproduce the muscles in a way that exactly correspond to anatomical science and often in the sculptures we can look at muscle groups portrayed in the act of participating in the movement of the body as a whole.

Art can therefore lead us, through its observation, to understand the anatomy represented or the reality of the cure; in fact, over the centuries, it has become a witness to illness, death, and healing activities. Through art it is possible to have evidence of missing pathologies and diseases useful for study, as the Italian doctor, G. Franceschini already wrote about it in 1906, who suggested that "Even the saddest and most painful sides of human life… have been… subject of study by artists, and how even the most pitiful and repulsive sciences of medicine have snatched from the creative brush of the passionate craftsman, pulsating works of life, truth, sentiment. And since beauty is the splendor of truth, it can be said that even the crudest truths of human pathology, clothed in the splendors of art from a skillful hand of craftsman, contributed to the creation of beauty, with sublime works of painting and sculpture" [1].

Art is also a representation of reality and can present itself as a "mirror" for the viewer who can understand emotions and knowledge of activities and meanings related to their personal experiences. Art, as experience, has often guided the pedagogical studies to be used as a tool to develop innovative educational methods [2].

The cognitive reaction to the processes of creation and use of the artistic image can determine knowledge. Starting from this concept, we can understand, first of all, why the observation of art can stimulate us to consider more than one interpretation and therefore more than one possible solution to a single question. Important in this area is the research of R. Arnheim. It explains the connection between visual perception and thought. "Identifying what we see is an act of knowledge," Arnheim tells us according to the psychology of perception [3]. When we look at

#### *Learning through Art in Medical Education DOI: http://dx.doi.org/10.5772/intechopen.101213*

something, mechanisms of understanding are rapidly implemented to recognize and grasp the sense of what is placed before our eyes. Furthermore, thanks to visual stimuli, thoughts and skills to solve problems are automatically set in motion. The careful observation of a work of art activates, in an almost instinctive way, multiple reasoning capable to achieve logical and analytical solutions, thanks to the multiple intelligences useful for cognitive development, including, mostly, the visual-spatial one [4].

In the care sector, there are many references to art as therapy and many studies that show psycho-physiological evidence of the positive effects that artistic practice and its use can have on the person for the promotion of well-being and health.

The role of visual arts and their usefulness, both for therapy and for the promotion of well-being and for the development of clinical skills, is highlighted by the report of the European section of the World Health Organization [5]. Furthermore, exposure to arts or exercising artistic activities can be "therapeutic," lowering cortisol levels and therefore limiting stress [6, 7].


**3.1 Algorithm for quantitative pedology (AQP)**

The AQP package allows to gather a set of functions to work and to analyze large soil profile collections. Depth functions of soil key properties used to distinguish the profiles are presented in **Figure 2** and **Figure 3**. The percentage values plotted along the profile shows the relative quantity of profiles used in the soil-depth function to calculate the statistics (median and quartiles) at each depth.

The study area presents shallow soils, sometimes with high base saturation; but the main limitation for agricultural use is the soil texture and high clay activity, which will influence the soil moisture to adequately manage the soils due to their high plasticity.

The pH and the calcium (Ca) content presented a linear trend of median values along depth (**Figure 2**), even though a smaller number of soil profiles (less than 25%) were used in the estimative for deeper than 150 cm depth. However, different patterns were shown for potassium (K), sodium (Na), electrical conductivity (EC), and cation exchange capacity (CEC). The soil key properties Ca, K, and CEC showed more variability around the median value, thus presenting a better predictive potential to distinguishing soils with different parent materials and high clay activity, respectively.

It is pertinent to select which soil key properties could indicate differences in soil behavior along depth and are relevant for land management. From the analyses of **Figure 3**, it is possible to conclude that clay and sand contents have an opposite and large variability among the profiles of the collection; thus, they have great potential to aid in the definition of soil management zones. Since both properties are

**Figure 2.** *Depth functions for soil key chemical properties. EC, electrical conductivity; CEC, cation exchange capacity.*

NDVI, b3/b7 ratio, and GSI (**Table 2**). These results do not agree with [44], who found no correlation between sand content and the Landsat 5 TM image data. Only the covariate b3/b7 was strongly correlated (r = 0.58 and 0.49, topsoil and subsoil, respectively) with the sand content [54], whereas the NDVI was moderately correlated (r = 0.39 and 0.36, topsoil and subsoil, respectively) and the other covariates weakly correlated, with r values below 0.27 (positive or negative)

*Pedometric Tools Applied to Zoning Management of Areas in Brazilian Semiarid Region*

*DOI: http://dx.doi.org/10.5772/intechopen.88526*

Inverse relationships were observed between clay content and the environmental covariates, but magnitudes were the same as those observed for sand (**Table 2**), except for the covariate b4 (topsoil) which had no correlation with sand; similar results were reported by [42, 55]. The most relevant covariates were b3/b7

(r = 0.56 and 0.51, topsoil and subsoil, respectively) and NDVI (r = 0.36 and 0.37, topsoil and subsoil, respectively). These results vary among authors in the literature. Significant correlations were obtained between clay, the NDVI index, and b3/b2 and b5/b7 band ratios by [44], while there was no correlation between clay and the b3/b7 ratio band. On the other hand, Ahmed and Iqbal [56] found significant correlations between clay and bands 4 and 6 using Landsat 5 TM.

The CEC was significantly correlated with the covariates b4, b5, b7, NDVI, and b3/b7 and b5/b7 ratios (**Table 2**). Only the covariate b3/b7 was strongly correlated (r = 0.45 and 0.44, topsoil and subsoil, respectively) with the CEC [54], whereas the other covariates were weakly correlated, with r values below 0.27 (positive or negative) (**Table 3**). A strong correlation was observed by [55] between the CEC and covariates measured by ASTER spectral bands (1–8). The Na content was significantly correlated with most of the covariates, except with b7 (topsoil) and b3/ b2 and b5/b7 ratios (**Table 2**). However, none of the covariates had a strong or moderate correlation with Na content (**Table 3**), which may explain the very low performance of the random forest model in the prediction of this soil property.

The study performed by Demattê et al. [57] highlighted that the correlation with

**Sand Clay CEC Na**

a particular spectral band is directly related with soil characteristics in specific

Depth (cm) 0–20 20–60 0–20 20–60 0–20 20–60 0–20 20–60 b1 0.06 0.07 0.07 0.03 0.06 0.03 0.19 0.32 b2 0.08 0.06 0.05 0.08 0.01 0.02 0.20 0.34 b3 0.06 0.05 0.03 0.06 0.05 0.02 0.19 0.32 b4 0.12 0.12 0.15 0.11 0.18 0.14 0.14 0.27 b5 0.23 0.20 0.25 0.19 0.24 0.20 0.10 0.20 b7 0.21 0.18 0.24 0.18 0.27 0.23 0.09 0.19 NDVI 0.39 0.36 0.36 0.37 0.22 0.23 0.21 0.27 B3/b2 0.07 0.07 0.02 0.06 0.13 0.11 0.10 0.12 b3/b7 0.58 0.49 0.56 0.51 0.45 0.44 0.24 0.29 b5/b7 0.02 0.00 0.06 0.02 0.21 0.20 0.03 0.10 GSI 0.27 0.26 0.21 0.24 0.03 0.04 0.15 0.23

*Pearson's correlation coefficient (r) between soil properties (sand, clay, CEC, and Na) and environmental*

regions, explaining the differences between study cases.

**Variables Soil properties**

*covariates (b1, b2, b3, b4, b5, b7, NDVI, b3/2, b3/b7, b5/b7, and GSI).*

(**Table 3**).

**Table 3.**

**47**

#### **Figure 3.** *Depth functions for soil key physical properties. PWP, permanent wilting point.*

important to irrigation and mechanization practices, they were selected as criteria for agricultural zoning.