Impact of Nitrogen Nutrition on Cannabis sativa: An Update on the Current Knowledge and Future Prospects
1Department of Biology, Complesso Universitario di Monte Sant’Angelo, University of Naples “Federico II”, Via Cinthia, I-80126 Napoli, Italy; simone.landi@unina.it (S.L.); giorgia.271291@gmail.com (G.C.)
2Department of Life Sciences, University of Siena, via P.A. Mattioli 4, I-53100 Siena, Italy; berni10@student.unisi.it
3Trees and Timber Institute-National Research Council of Italy (CNR-IVALSA), via Aurelia 49, 58022 Follonica (GR), Italy
4Environmental Research and Innovation Department, Luxembourg Institute of Science and Technology, 5, rue Bommel, Z.A.E. Robert Steichen, L-4940 Hautcharage, Luxembourg; jean-francois.hausman@list.lu
*Correspondence: gea.guerriero@list.lu (G.G.); sergio.esposito@unina.it (S.E.)Abstract
Nitrogen (N) availability represents one of the most critical factors affecting cultivated crops. N is indeed a crucial macronutrient influencing major aspects, from plant development to productivity and final yield of lignocellulosic biomass, as well as content of bioactive molecules. N metabolism is fundamental as it is at the crossroad between primary and secondary metabolic pathways: Besides affecting the synthesis of fundamental macromolecules, such as nucleic acids and proteins, N is needed for other types of molecules intervening in the response to exogenous stresses, e.g. alkaloids and glucosinolates. By partaking in the synthesis of phenylalanine, N also directly impacts a central plant metabolic ‘hub’—the phenylpropanoid pathway—from which important classes of molecules are formed, notably monolignols, flavonoids and other types of polyphenols. In this review, an updated analysis is provided on the impact that N has on the multipurpose crop hemp (Cannabis sativa L.) due to its renewed interest as a multipurpose crop able to satisfy the needs of a bioeconomy. The hemp stalk provides both woody and cellulosic fibers used in construction and for biocomposites; different organs (leaves/flowers/roots) are sources of added-value secondary metabolites, namely cannabinoids, terpenes, flavonoids, and lignanamides. We survey the available literature data on the impact of N in hemp and highlight the importance of studying those genes responding to both N nutrition and abiotic stresses. Available hemp transcriptomic datasets obtained on plants subjected to salt and drought are here analyzed using Gene Ontology (GO) categories related to N metabolism. The ultimate goal is to shed light on interesting candidate genes that can be further studied in hemp varieties growing under different N feeding conditions and showing high biomass yield and secondary metabolite production, even under salinity and drought.
1. Introduction
In recent years, hemp (Cannabis sativa L.) has been the object of research due to its potential as crop with multiple uses in a required bio-safe agriculture and economy [1,2,3]. Hemp is one of the earliest domesticated crops [4] and is commonly used in over 25,000 commercial products [5]. Industrial hemp differs from psychotropic varieties in the tetrahydrocannabinol (THC) content which is <0.3% and it is cultivated both for seed oil and fibers [6]. The cortex of the hemp stalk contains highly crystalline cellulosic fibers, known as bast fibers, which mechanically support the phloem. These fibers are strong and long and find application as green substitutes of synthetic fibers in biocomposites.
Hemp is polyvalent in terms of applications, as it also produces interesting secondary metabolites: The industrial varieties contain cannabidiol (CBD) [7], but also other interesting compounds from a pharmaceutical point of view, such as specific terpenes, responsible for the typical scent. Considering these medical and industrial applications, nowadays hemp is extensively cultivated in almost 50 countries [2,3,8,9]. Interestingly, among the 16 best producers, 11 are European countries (e.g., France, Austria, Italy, among others) [3]. The production of hemp tow waste and seeds constantly increased worldwide from 1997–2007; then a reduction was observed until 2010. In the last decade, an increase was again recorded, indicating European Union (EU) countries as the best producers both for hemp seeds and—in the recent years— tow waste, overtaking China (Figure 1).
Data about harvested area reflect the production: American countries showed a lower production, resulting in USA as the major hemp importer worldwide [5].
This reignited the interest of USA in hemp cultivation, obviously induced by an increased market demand. The major hemp products, namely fiber and CBD, showed market prices ranging from 4848 $/Ha of fiber to the 25000 $/Ha of CDB [3]. This directed the scientific research to improve the knowledge about stem development, genetic regulation of fiber traits, secondary metabolites, biosynthetic pathways and their engineering, increase of grain yield, oil composition, and response to biotic and abiotic stresses [3].
The availability of N affects the response of plants to exogenous cues; therefore, understanding the regulation of N metabolism under stress is crucial for agriculture. N represents indeed a key macronutrient for plant cells and its availability influences major aspects of plant physiology, such as photosynthesis, development, growth, flowering, and senescence [10,11].
Nevertheless, despite the agro-economic interest for hemp, many aspects affecting the productivity of this crop remain still poorly studied. N availability is an example: Its impact on hemp growing under abiotic stress conditions is even less understood [3]. Besides the impact on plant biomass accumulation, mineral nutrition can also affect the production of secondary metabolites, as for example shown for the cannabinoid metabolism [12].
Hemp shows a number of interesting agricultural features, such as low nitrogen (N) input requirement [2,13,14,15], heat tolerance [2,15], phytoremediation [16,17], positive effects on environments [18], benefits in crop rotation [19] and availability of high-yield varieties [20,21].
Hemp production is particularly threatened by abiotic stresses and N starvation, the major constraints limiting crop yield [2,21,22]; it must be underlined that these two factors are strictly correlated, severely impacting cell metabolism, plant growth and differentiation [23]. Abiotic stresses induce a number of morphological, physiological and molecular changes affecting plant growth and productivity [10]; this is particularly true in hemp, where photosynthesis-related pathways and associated genes are strongly downregulated [24]. Drought and salinity seriously threaten agricultural productivity. Important effects are observed in the stem tissues of salt-stressed hemp: Besides the presence of smaller xylem vessels [25], a decrease in the number of bast fibers is observed (Figure 2). This is particularly evident for secondary bast fibers (Figure 2, dotted box). Environmental stress significantly affects two important feature of hemp commercial varieties, i.e., stem and seed yield. The relationship between yield and environmental constraints was due to flowering time, mainly regulated by photoperiod and temperature [15].
Humic acids together with macronutrients—such as N, K, and P—were shown to affect the cannabinoid profile; this effect is depending on plant organs and acts in a spatial-dependent manner (e.g., top-middle-bottom of the plant) [12]: For example, humic acids reduced the variability in cannabinoid abundance in the different organs (flowers, leaves, inflorescences), but this increased homogeneity was accompanied by a decrease in the upper regions of plants, which normally contain high levels of these secondary metabolites.
In light of the central role of N as macronutrient affecting both primary and secondary plant metabolic pathways, we here provide an overview of the current knowledge on hemp physiology in relation with N nutrition. We also report genes related to N metabolism and responding to salt/drought stress in hemp to pave the way to future strategies improving specific traits under unfavorable conditions for this economically important multipurpose crop.
2. Physiological Effects of N Availability in Hemp
The availability of macronutrients in the soil heavily affects plant growth and development [10,11]. When soils are depleted of particular nutrients, severe limitations in biomass production occur [11]. These effects are particularly true when N nutrition is considered, due to the essential role of N in the structure of crucial molecules for life, such as amino acids and N-bases. Therefore, it is not unexpected that plants evolved a number of mechanisms to avoid severe damage when N is limiting in the soil [11]. On the other hand, it is not rare that plants can experience a prolonged limitation of N; when this occurs, recycling mechanisms have been developed to avoid—at least in part—growth stop and/or limiting stress symptoms, in order to guarantee plant survival [26]. Nutrient starvation may cause an important physiological process, nutrient resorption; this parameter contributes to nutrient retention and it is intended as a strategy for nutrient storage [26,27]. Nutrient resorption is defined as the percentage of a nutrient stored by a plant before the beginning of the senescence process (physiological or stress-induced) and the resorption proficiency as the final concentration of a specific nutrient in tissues after senescence [26]. Resorption process allows the recycling primarily of N and P and it is intuitive that plants living in nutrient-limiting soils or in stress environments (e.g. arid ecosystems) show better resorption capabilities [27]. This aspect was investigated in hemp in comparison with other important species in semi-arid environments ([28], summarized in Table 1). Hemp yield is limited by nutrient availability and this is particularly true in semi-arid ecosystems, where drought conditions are often accompanied by N deficiency [2,21]. Interestingly, N resorption efficiency (NRE) showed a general decrease in Cannabis plants in response to increased soil N availability. Under control conditions, hemp showed the best NRE value as compared to Artemisia scoparia, Chenopodium acuminatum and Phragmites communis. Furthermore, hemp showed a high NRE in the absence of N and upon N- and phosphorus (P)-enriched environments. Less difference was reported between the analyzed species for PRE (phosphorus resorption efficiency) [28].
The effects of N supply on photosynthetic N-use efficiency of plant canopy (PNUEc) are determined by the effect of N on the size of canopy and/or leaf area index (LAI). PNUEc of hemp increased with decreasing N fertilization and this is correlated with a reduction in LAI [2]. Furthermore, these effects could be correlated to a variation in the absolute amount of the specific leaf N content (SLN). Interestingly, upon water scarcity, hemp showed a concomitant decrease of PNUEc value and increase of canopy photosynthetic water use efficiency (PWUEc). This parameter showed no or less differences under varying N inputs [2].
Experiments carried out in Eastern Canada (Québec) revealed the effects of N, P, and K fertilization on the biomass and seed yield in two hemp cultivars, CRS-1 and Anka [29]. The results revealed the existence of a strong interaction environment x fertilization, as well as a dependence on the cultivar. A more than two-fold seed yield was obtained with 200 kg N/ha; an effect was observed on cellulose and hemicellulose content as well, but it was minimal. The final recommendation was to use N fertilization >200 kg N/ha, which is higher than the level normally used in Western Canada, i.e., 150 kg N/ha [29]. This study indicates the importance of evaluating the geographical location and relative environmental conditions (e.g., Eastern Canada is more humid than Western Canada) before establishing agronomic recommendations relative to N fertilization of hemp cultures.
Another study evaluated the impact of N fertilization on photosynthesis, fibers and seed oil content in a cultivar of hemp grown in Latvia [30]. High doses of N (100 kg/ha and in the form of NH4NO3) increased the content of chlorophyll already 7 days after the first application and improved the Performance Index (PI) by increasing photosystem II activity. Although the height of plants increased by 11% with N, the fiber yield was ca. 8% lower than non-fertilized plants: this indicates a carbon-dependent assimilation of nitrate into amino acids which lowers carbohydrate biosynthesis. High N can indeed affect the mechanical strength of stems and increase lodging by reducing both cellulose and lignin contents [31]. The composition of seed oil did not change significantly upon different fertilizations, although a general decrease was observed, as seen with fibers, a finding suggesting a preferential metabolic shunt towards amino acid and protein biosynthesis.
It is worth mentioning here the beneficial effect of plant growth promoting bacteria (PGPB) on growth and development: PGPB indeed improve crop yield by enhancing nutrient mobilization and protecting against exogenous stresses [32]. Beneficial bacteria greatly increase the root surface area, thereby improving nutrients’ uptake from the soil; some bacteria can fix N, thus improving the fitness of plants in N-limited environments; other micro-organisms are able to solubilize P salts and thus allow access to otherwise recalcitrant forms [33]. A recent perspective article discussed the interest of testing PGPB on hemp [34] as biostimulants; it was shown that PGPB clearly improve nutrient use, tolerance to (a)biotic stresses, and accumulation of phytochemicals. Treatment of C. sativa with the biostimulant Mammoth P™ improved growth, but a higher amount of cannabinoids could not be detected [35].
5. Breeding Strategies in Hemp: Nutrient Management and Synthesis of Secondary Metabolites
The renewed interest in hemp cultivation encouraged efforts in hemp breeding programs in the last 15 years [73,74]. These programs were finalized to obtain a better fiber quality, an increased yield andsynthesis of secondary metabolites, as well as to improve and control the time of flowering [75]. Breeding programs were performed by using various strategies, such as mass selection, cross, and hybrid breeding and using advanced technologies such as next-generation sequencing (NGS), genotype by sequencing (GBS), use for genetic maps [73,75,76]. Furthermore, breeding strategies and polyploidization induction also improve the production of secondary metabolites [77]. Large-scale rearrangements or duplications of genome enable new allelic combinations, by increasing the genetic variety and conferring physiological advantages in particular lineages [77]. The manipulation of ploidy status is a valuable and recognized tool for plant breeding that can induce the development of larger organs, by increasing heterozygosity, hybrid vigor; furthermore, this strategy can often be linked to an increased tolerance to stress [78]. A number of studies were recently reported about polyploidization in hemp [79,80,81], which naturally occurs as a diploid plant (2n = 20) [8]. Recently, the induction of polyploidy in C. sativa caused reduced CO2 fixation and carbohydrate content; an increase in flavonoid levels, possibly related to UV defense; reduction in cellulose content [79]. This was accompanied by an enhanced ability in nutrient uptake, thus highlighting an increased tolerance against starvation [79]. Mixoploid vegetative plants showed a higher content of Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) [79].
N and P were shown to be essential in species with large genomes [82]. A study on 96-hexaploid wheat accessions reported an increased ability of N uptake in a polyploid population, suggesting this as an effective strategy to identify favorable QTLs for marker-assisted breeding programs [83].
It is worth noting that nutrient uptake and transport are key aspects in hemp, regulating the transition from vegetative to reproductive stage. The genes modulating this transition are thus interesting candidates for breeding to improve bast fiber quality [84].
The possibility to grow hemp in adverse environments is of high interest for breeding [75,85]. Specific cultivars of hemp have been selected for cultivation in specific countries and/or environments (e.g., Italy, France, Russia, China, USA), showing specific adaptations to low temperatures, drought, nutrient starvation and salinity [75]; furthermore, varieties showing phytoremediation potential have been obtained [86]. The relationship between nutrient availability and marketable products of hemp is a major topic for hemp breeding; particularly, the effects of N on hemp fibers are complex. N availability could influence hemp growth, plant harvesting, biomass production and fiber yield by impacting primary metabolism and the provision of precursors needed for cell wall synthesis [87].
6. Manipulation of the Cannabinoid Biosynthetic Pathway and Their Relation with N
Secondary metabolites, such as phytocannabinoids have application in the pharma industry, as well as cosmetics and the formulation of insecticides and antibacterial products [3,5,7,88,89,90]. Phytocannabinoids, such as THC and CBD, are secondary metabolites produced by hemp. THC is responsible for the psychoactive properties of hemp, whereas CBD is used against pain, anxiety, depression, and sleep disorders [6,91]. THC, cannabinol (CBN), cannabigerol (CBG) and CBD levels were studied in presence of N, P and potassium (K) supplementation in hemp cultivars grown for medical applications [12]. Interestingly, the concomitant N, P, and K supplementation showed physiological changes and modifications in the phytocannabinoid content. Fan leaf biomass increased with NPK and the addition of these elements also induced an increase in flower and stem biomass. These changes were accompanied by modified phytocannabinoid contents. THC levels decreased by 19% in inflorescence leaves of plants treated with NPK, while CBG increased by 71% in flowers. CBN levels decreased in flowers and inflorescence leaves by 38% and 36%, respectively, in the presence of NPK [12].
The major cannabinoids THC and CBD are both synthesized as acid forms from a common precursor, cannabigerolic acid (CBGA), by two different enzymes: THCA synthase (THCAS) and CBDA synthase (CBDAS) [92,93]. It has been proposed that THCAS and CBDAS are closely linked to each other, so that one of the two can be inactivated in drug- or hemp producing strains, respectively [80]. Tetraploid Cannabis strains producing enhanced THC and TCH/CDB levels showed phenotypes with larger leaves, increased density of trichomes and stomata and a rearrangement of secondary metabolites’ profile [81].
Recently, the enzymes responsible for the synthesis of cannabinoids (tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase) were found to be secreted in trichomes’ exudates which is rich in hydrophilic, amphiphilic and osmoprotective metabolites providing the right micro-environment for the enzyme solubility and catalytic activity [94].
The first enzymes of the cannabinoid biosynthetic pathways were described and characterized [95,96]. Similarly, fatty acids and isoprenoids were originally identified as cannabinoid precursors [97]; particularly, hexanoyl-CoA and malonyl-CoA were recognized as critical compounds in the cannabinoid pathway. Using a combination of transcriptomic and mass spectrometry approaches in flowers from female hemp, node enzymes for the initial steps of cannabinoid biosynthetic pathway were identified. The enzyme responsible for the aldolic condensation between hexanoyl-CoA with three molecules of malonyl-CoA was identified by Gagne et al. [95]. This is the olivetolic acid cyclase (OAC), which catalyzes a C2–C7 intramolecular aldol condensation with carboxylate retention to form olivetolic acid. Similarly, CsAAE1 and CsAAE2 (acyl activating enzymes) were identified as major suppliers of hexanoyl-CoA [96].
These results were recently used to reproduce a complete cannabinoid biosynthetic route in yeast. This engineered pathway was reconstructed in Saccharomyces cerevisiae from galactose, by triggering a flux from the mevalonate pathway to geranyl pyrophosphate and by overexpressing olivetolic acid cyclase together with a geranylpyrophosphate:olivetolate geranyltransferase [98].
7. Conclusions
In this review, we illustrated how abiotic stresses and N metabolism are strictly interdependent in C. sativa. The relationship between nutrient availability and marketable products of hemp is a major topic for hemp breeding; in particular, N availability could influence hemp growth, plant harvesting, biomass production and fiber yield.
The results discussed strongly support the idea of a co-regulation of nitrate transporters, N metabolism-related genes, transcription factors and genes involved in secondary metabolism as part of a complex machinery engaged by hemp to counteract abiotic stress. Therefore, these gene families could represent potential targets for genetic improvement in hemp, as well as other fiber crops, ameliorating abiotic stress tolerance, nitrogen assimilation and, ultimately, production of biomass and secondary metabolites.
Acknowledgments
The authors thank Laurent Solinhac for the help with the microscope. G.G. and J.-F.H. acknowledge the Fonds National de la Recherche, Luxembourg, (Project CANCAN C13/SR/5774202).
Funding
This research received no funding.
Conflicts of Interest
The authors declare no conflict of interest.
| Plant | Control | N Supply | P Supply | N + P Addition | ||||
|---|---|---|---|---|---|---|---|---|
| NRE (%) | PRE (%) | NRE (%) | PRE (%) | NRE (%) | PRE (%) | NRE (%) | PRE (%) | |
| C. sativa | 62 | 42 | 35 | 35 | 52 | 38 | 25 | 41 |
| P. communis | 42 | 10 | 45 | 20 | 58 | 10 | 60 | 15 |
| A. scoparia | 42 | 50 | 20 | 62 | 55 | 40 | 22 | 50 |
| C. acuminatum | 48 | 45 | 30 | 60 | 40 | 42 | 18 | 58 |
| Locus | Behavior vs. Abiotic Stress | Annotation | GO or KEGG Categories Related to N Metabolism |
|---|---|---|---|
| gi_351617093_gb_JP471394.1 | UP upon Drought | MYBR domain class transcription factor | GO:0010243 |
| gi_351606916_gb_JP461241.1 | UP upon Drought | WRKY transcription factor 33-like | GO:0010243 |
| gi_351617961_gb_JP472262.1 | UP upon Drought | Vinorine synthase-like | GO:0006807 |
| gi_351624360_gb_JP478661.1 | UP upon Drought | Triacylglycerol lipase | GO:0006807 |
| gi_351627864_gb_JP480747.1 | UP upon Drought | Vinorine synthase-like | GO:0034641 |
| gi_351612890_gb_JP467191.1 | UP upon Drought | Transcription factor LHY | GO:0010243 |
| gi_351603990_gb_JP458344.1 | UP upon Drought | Cysteine-rich receptor-like protein kinase | GO:0071705 |
| gi_351624708_gb_JP479009.1 | UP upon Drought | Delta-1-pyrroline-5-carboxylate synthetase | GO:0034641 |
| gi_351618788_gb_JP473089.1 | UP upon Drought | Pleiotropic drug resistance protein | GO:0006995 |
| gi_351599092_gb_JP453596.1 | UP upon Drought | ACD1-like | GO:0044270 |
| gi_351596616_gb_JP451172.1 | UP upon Drought | Probable peptide/nitrate transporter | GO:0006807 |
| gi_351625252_gb_JP479553.1 | UP upon Drought | Glucose-methanol-choline oxidoreductase | GO:0006807 |
| gi_351629105_gb_JP481988.1 | UP upon Drought | Hypothetical protein | GO:0034641 |
| gi_351628557_gb_JP481440.1 | UP upon Drought | Deoxytaxol N-benzoyltransferase | GO:0006807 |
| gi_351622676_gb_JP476977.1 | UP upon Drought | Shikimate O-hydroxycinnamoyltransferase | GO:0034641 |
| gi_351597997_gb_JP452531.1 | UP upon Drought | Aldehyde dehydrogenase | GO:0006807 |
| gi_351624288_gb_JP478589.1 | UP upon Drought | Hypothetical protein | GO:0051171 |
| gi_351615767_gb_JP470068.1 | UP upon Drought | MYB domain protein 20 | GO:0071705 |
| gi_351623654_gb_JP477955.1 | DOWN upon Drought | 21 kDa protein | GO:0071705 |
| gi_351629055_gb_JP481938.1 | DOWN upon Drought | Urea-proton symporter | GO:0006995 |
| gi_351624448_gb_JP478749.1 | DOWN upon Drought | Allantoinase | GO:0006995 |
| gi_351624658_gb_JP478959.1 | DOWN upon Drought | Salutaridinol 7-O-acetyltransferase | GO:0006807 |
| gi_351597746_gb_JP452287.1 | DOWN upon Drought | Anthranilate N-benzoyltransferase | GO:0006807 |
| gi_351605867_gb_JP460208.1 | DOWN upon Drought | Nitrate transporter 1.2 | GO:0006807 |
| gi_351602034_gb_JP456469.1 | DOWN upon Drought | Nitrate transporter 1.2 | GO:0006807 |
| gi_351619289_gb_JP473590.1 | DOWN upon Drought | Non-symbiotic hemoglobin 2 | GO:0009399 |
| gi_351591331_gb_JP449779.1 | DOWN upon Drought | Protein PHR1-LIKE 1 | GO:0034641 |
| gi_351625347_gb_JP479648.1 | DOWN upon Drought | Pleiotropic drug resistance protein 2 | GO:0006995 |
| gi_351590806_gb_JP449264.1 | DOWN upon Drought | Vacuolar iron transporter | GO:0009399 |
| gi_351598838_gb_JP453346.1 | DOWN upon Drought | Pleiotropic drug resistance protein | GO:0006995 |
| gi_351620563_gb_JP474864.1 | DOWN upon Drought | Leucine-rich repeat family protein | GO:0006995 |
| gi_351597483_gb_JP452028.1 | DOWN upon Drought | LRR receptor-like serine/threonine kinase | GO:0071705 |
| gi_351596259_gb_JP450816.1 | DOWN upon Drought | Glutamine synthetase leaf isozyme | GO:0009399/Ko00910 |
| gi_351624862_gb_JP479163.1 | DOWN upon Drought | LRR receptor-like serine/threonine kinase | GO:0071705 |
| gi_351624507_gb_JP478808.1 | DOWN upon Drought | L-3-cyanoalanine synthase 1 | GO:0051410 |
| gi_351623596_gb_JP477897.1 | DOWN upon Drought | Tropinone reductase | GO:0044271 |
| gi_351605608_gb_JP459952.1 | DOWN upon Drought | Major facilitator superfamily protein | GO:0006807 |
| gi_351606133_gb_JP460466.1 | DOWN upon Drought | BAHD acyltransferase | GO:0006807 |
| gi_351623568_gb_JP477869.1 | DOWN upon Drought | Tocopherol O-methyltransferase | GO:0034641 |
| gi_351601188_gb_JP455639.1 | DOWN upon Drought | LRR receptor-like serine/threonine kinase | GO:0071705 |
| gi_351598272_gb_JP452798.1 | DOWN upon Drought | Aminomethyltransferase, mitochondrial | ko00910 |
| gi_351612051_gb_JP466352.1 | DOWN upon Drought | Carbonic anhydrase 2 | ko00910 |
| gi_351615730_gb_JP470031.1 | DOWN upon Drought | Carbonic anhydrase, chloroplastic | ko00910 |
| gi_351617853_gb_JP472154.1 | DOWN upon Drought | Carbonic anhydrase, chloroplastic | ko00910 |
| gi_351621906_gb_JP476207.1 | DOWN upon Drought | Bifunctional monodehydroascorbate reductase | ko00910 |
| PK00197.1 | UP upon Salinity | Glutamate dehydrogenase | ko00910 |
| PK06425.1 | UP upon Salinity | Glutamate synthase | ko00910 |
| PK21222.1 | DOWN upon Salinity | Carbonic anhydrase 1 | ko00910 |