Revealing floral metabolite network in tuberose that underpins scent volatiles synthesis, storage and emission.

KEY MESSAGE: The role of central carbon metabolism in the synthesis and emission of scent volatiles in tuberose flowers was revealed through measurement of changes in transcripts and metabolites levels. Tuberose or Agave amica (Medikus) Thiede & Govaerts is a widely cultivated ornamental plant in several subtropical countries. Little is known about metabolite networking involved in biosynthesis of specialized metabolites utilizing primary metabolites. In this study, metabolite profiling and gene expression analyses were carried out from six stages of maturation throughout floral lifespan. Multivariate analysis indicated distinction between early and late maturation stages. Further, the roles of sugars viz. sucrose, glucose and fructose in synthesis, glycosylation and emission of floral scent volatiles were studied. Transcript levels of an ABC G family transporter (picked up from the floral transcriptome) was in synchronization with terpene volatiles emission during the anthesis stage. A diversion from phenylpropanoid/benzenoid to flavonoid metabolism was observed as flowers mature. Further, it was suggested that this metabolic shift could be mediated by isoforms of 4-Coumarate-CoA ligase along with Myb308 transcription factor. Maximum glycosylation of floral scent volatiles was shown to occur at the late mature stage when emission declined, facilitating both storage and export from the floral tissues. Thus, this study provides an insight into floral scent volatiles synthesis, storage and emission by measuring changes at transcripts and metabolites levels in tuberose throughout floral lifespan.

Calm Before the Storm: A Glimpse into the Secondary Metabolism of Aspergillus welwitschiae, the Etiologic Agent of the Sisal Bole Rot.

Aspergillus welwitschiae is a species of the Nigri section of the genus Aspergillus. In nature, it is usually a saprotroph, decomposing plant material. However, it causes the bole rot disease of Agave sisalana (sisal), a plant species used for the extraction of hard natural fibers, causing great economic loss to this culture. In this study, we isolated and sequenced one genome of A. welwitschiae (isolate CCMB 674 (Collection of Cultures of Microorganisms of Bahia)) from the stem tissues of sisal and performed in silico and wet lab experimental strategies to describe its ability to produce mycotoxins. CCMB 674 possesses 64 secondary metabolite gene clusters (SMGCs) and, under normal conditions, it produces secondary metabolism compounds that could disturb the cellular cycle of sisal or induce abnormalities in plant growth, such as malformin C. This isolate also produces a pigment that might explain the characteristic red color of the affected tissues. Additionally, this isolate is defective for the production of fumonisin B1, and, despite bearing the full cluster for the synthesis of this compound, it did not produce ochratoxin A. Altogether, these results provide new information on possible strategies used by the fungi during the sisal bole rot, helping to better understand this disease and how to control it.

Identification and Expression of SAUR Genes in the CAM Plant Agave.

Agave species are important crassulacean acid metabolism (CAM) plants and widely cultivated in tropical areas for producing tequila spirit and fiber. The hybrid H11648 of Agave ((A. amaniensis × A. angustifolia) × A. amaniensis) is the main cultivar for fiber production in Brazil, China, and African countries. Small Auxin Up-regulated RNA (SAUR) genes have broad effect on auxin signaling-regulated plant growth and development, while only few SAUR genes have been reported in Agave species. In this study, we identified 43, 60, 24, and 21 SAUR genes with full-length coding regions in A. deserti, A. tequilana, A. H11648, and A. americana, respectively. Although phylogenetic analysis revealed that rice contained a species-specific expansion pattern of SAUR gene, no similar phenomena were observed in Agave species. The in silico expression indicated that SAUR genes had a distinct expression pattern in A. H11648 compared with other Agave species; and four SAUR genes were differentially expressed during CAM diel cycle in A. americana. Additionally, an expression analysis was conducted to estimate SAUR gene expression during different leaf developmental stages, abiotic and biotic stresses in A. H11648. Together, we first characterized the SAUR genes of Agave based on previously published transcriptome datasets and emphasized the potential functions of SAUR genes in Agave's leaf development and stress responses. The identification of which further expands our understanding on auxin signaling-regulated plant growth and development in Agave species.

Phosphate-solubilizing bacteria improve Agave angustifolia Haw. growth under field conditions.

BACKGROUND: Phosphate-solubilizing bacteria (PSB) can be an environment-friendly strategy to improve crop production in low-phosphorus (P) or P-deficient soils. The effect of indigenous mixed inocula of PSB on Agave angustifolia Haw. growth was assessed. The four treatments evaluated were T1 (Pseudomonas luteola + Enterobacter sp.), T2 (Pseudomonas luteola + Bacillus sp.), T3 (Pseudomonas luteola + Acinetobacter sp.), and T4 (control); each was replicated 25 times using a completely randomized design during 12 months under rain-fed conditions. Additionally, P solubilization in vitro of the mixed inocula with three different sources of inorganic P was tested. RESULTS: The mixed inocula were able to solubilize more P from tricalcium phosphate Ca3 (PO4 )2 than from aluminum phosphate (AlPO4 ) and iron phosphate (FePO4 ). Relative to the control, T2 increased plant height by 22.9%, leaf dry weight by 391.4%, plant stem diameter by 49.6%, and root dry weight by 193.9%. The stem solid soluble content increased 50.0% with T1. Plant-available soil P increased 94.6% with T3 and 77.3% with T1. Soil alkaline phosphatase activity increased 85.9% with T1. CONCLUSION: T2 was the mixed inoculum that most improved Agave angustifolia plant growth. The indigenous mixed inocula of PSB evaluated appears to be a practical and efficient option for promoting field growth of Agave angustifolia plants. However, further research is necessary to achieve a deeper understanding of the relationships between different PSB species and their effects on agave, which may reveal some of the mechanisms of the synergistic interactions that are involved in the promotion of plant growth. © 2019 Society of Chemical Industry.

Transcriptome analysis of bolting in A. tequilana reveals roles for florigen, MADS, fructans and gibberellins.

BACKGROUND: Reliable indicators for the onset of flowering are not available for most perennial monocarpic species, representing a drawback for crops such as bamboo, agave and banana. The ability to predict and control the transition to the reproductive stage in A. tequilana would represent an advantage for field management of agaves for tequila production and for the development of a laboratory model for agave species. RESULTS: Consistent morphological features could not be determined for the vegetative to reproductive transition in A. tequilana. However, changes in carbohydrate metabolism where sucrose decreased and fructans of higher degree of polymerization increased in leaves before and after the vegetative to reproductive transition were observed. At the molecular level, transcriptome analysis from leaf and shoot apical meristem tissue of A. tequilana plants from different developmental stages identified OASES as the most effective assembly program and revealed evidence for incomplete transcript processing in the highly redundant assembly obtained. Gene ontology analysis uncovered enrichment for terms associated with carbohydrate and hormone metabolism and detailed analysis of expression patterns for individual genes revealed roles for specific Flowering locus T (florigen), MADS box proteins, gibberellins and fructans in the transition to flowering. CONCLUSIONS: Based on the data obtained, a preliminary model was developed to describe the regulatory mechanisms underlying the initiation of flowering in A. tequilana. Identification of specific promoter and repressor Flowering Locus T and MADS box genes facilitates functional analysis and the development of strategies to modulate the vegetative to reproductive transition in A. tequilana.

Cytosolic calcium localization and dynamics during early endosperm development in the genus Agave (Asparagales, Asparagaceae).

Calcium is a secondary messenger that regulates and coordinates the cellular responses to environmental cues. Despite calcium being a key player during fertilization in plants, little is known about its role during the development of the endosperm. For this reason, the distribution, abundance, and dynamics of cytosolic calcium during the first stages of endosperm development of Agave tequilana and Agave salmiana were analyzed. Cytosolic calcium and actin filaments detected in the embryo sacs of Agave tequilana and A. salmiana revealed that they play an important role during the division and nuclear migration of the endosperm. After fertilization, a relatively high concentration of cytosolic calcium was located in the primary nucleus of the endosperm, as well as around migrating nuclei during the development of the endosperm. Cytosolic calcium participates actively during the first mitosis of the endosperm mother cell and interacts with the actin filaments that generate the motor forces during the migration of the nuclei through the large cytoplasm of the central cell.

A model of environmental limitations on production of Agave americana L. grown as a biofuel crop in semi-arid regions.

Plants that use crassulacean acid metabolism (CAM) have the potential to meet growing agricultural resource demands using land that is considered unsuitable for many common crop species. Agave americana L., an obligate CAM plant, has potential as an advanced biofuel crop in water-limited regions, and has greater cold tolerance than other high-yielding CAM species, but physiological tolerances have not been completely resolved. We developed a model to estimate the growth responses of A. americana to water input, temperature, and photosynthetically active radiation (PAR). The photosynthetic response to PAR was determined experimentally by measuring the integrated leaf gas exchange over 24 h after acclimation to six light levels. Maximum CO2 fixation rates were observed at a PAR intensity of 1250 µmol photons m-2 s-1. Growth responses of A. americana to water and temperature were also determined, and a monthly environmental productivity index (EPI) was derived that can be used to predict biomass growth. The EPI was calculated as the product of water, temperature, and light indices estimated for conditions at a site in Maricopa (Arizona), and compared with measured biomass at the same site (where the first field trial of A. americana as a crop was completed). The monthly EPI summed over the lifetime of multi-year crops was highly correlated with the average measured biomass of healthy 2- and 3-year-old plants grown in the field. The resulting relationship between EPI and biomass provides a simple model for estimating the production of A. americana at a monthly time step according to light, temperature, and precipitation inputs, and is a useful tool for projecting the potential geographic range of this obligate CAM species in future climatic conditions.

Smells from the desert: Microbial volatiles that affect plant growth and development of native and non-native plant species.

The plant microbiota can affect host fitness via the emission of microbial volatile organic compounds (mVOCs) that influence growth and development. However, evidence of these molecules and their effects in plants from arid ecosystems is limited. We screened the mVOCs produced by 40 core and representative members of the microbiome of agaves and cacti in their interaction with Arabidopsis thaliana and Nicotiana benthamiana. We used SPME-GC-MS to characterize the chemical diversity of mVOCs and tested the effects of selected compounds on growth and development of model and host plants. Our study revealed that approximately 90% of the bacterial strains promoted plant growth both in A. thaliana and N. benthamiana. Bacterial VOCs were mainly composed of esters, alcohols, and S-containing compounds with 25% of them not previously characterized. Remarkably, ethyl isovalerate, isoamyl acetate, 3-methyl-1-butanol, benzyl alcohol, 2-phenylethyl alcohol, and 3-(methylthio)-1-propanol, and some of their mixtures, displayed beneficial effects in A. thaliana and also improved growth and development of Agave tequilana and Agave salmiana in just 60 days. Volatiles produced by bacteria isolated from agaves and cacti are promising molecules for the sustainable production of crops in arid and semi-arid regions.

Starch accumulation is associated with active growth in A. tequilana.

Transcriptome analysis of different tissues and developmental stages of A. tequilana plants led to the identification of full length cDNAs and the corresponding amino acid sequences for enzymes involved in starch metabolism in this species. Comparison with sequences from other species confirmed the identities of putative A. tequilana starch metabolism genes and uncovered differences in the evolutionary patterns of these genes between gramineous and non-gramineous monocotyledons. In silico expression patterns showed high levels of expression of starch metabolism genes in shoot apical meristem tissue and histological studies showed the presence of starch in leaf primordia surrounding the shoot apical meristem and in the primary thickening meristem of the stem. Starch was also found to accumulate significantly in developing floral organs and immature embryos. Low levels of starch were observed overall in leaf tissue with the exception of stomatal guard cells where starch was abundant. In root tissue, starch was only observed in statoliths at the root tip. A. tequilana starch grains were found to be small in comparison to other species and have an almost spherical form. The data for gene expression and histological localization are consistent with a role for starch as a transient carbohydrate store for actively growing tissues in A. tequilana.

Steroidal Saponin and Flavonol Content and Antioxidant Activity during Sporophyte Development of Maguey (Agave salmiana).

Agave salmiana Otto ex Salm-Dyck has traditionally been used for the production of fermented beverage known as "pulque" that has recently gained acceptance as a functional food. However, the plant requires up to 10 years to be used as raw material. The objective of this work was to evaluate the antioxidant and bioactive principles of Agave salmiana during different stages of development. Wild grown plants from Coahuila, Mexico, were identified based on leaf and spine traits to obtain a representative sample from six different stages of development (I-VI) from 1 to 7 years old. Total phenolic content (TPC), antioxidant activity (AOX), as well as composition and content of flavonols and saponins by HPLC-MS-TOF and HPLC-ELSD-PDA were evaluated. Concentrations of TPC were found to be between 5 to 13 mg gallic acid equivalents/g, reaching a maximum at stage II. The AOX presented a negative tendency from stage I to stage VI (from 148 to 50 µmol Trolox equivalents/g respectively). Kaempferol, quercetin and five saponins were identified. Similar to AOX, flavonols presented a negative concentration tendency with a reduction of 65% between the stage I and VI. Plants of stage III and IV presented the highest content of saponins, particularly chlorogenin glycoside, containing 3.19 and 2.90 mg protodioscin equivalents/g, respectively. These data suggest that plants from stages I to IV may be used as a source of antioxidant and bioactive principles, and that the content of these metabolites could be used as a marker to determine the developmental stage of the plant.

Micropropagation of Agave Species.

The genus Agave originates from the American continent and grows in arid and semiarid places, being México the center of origin. Many species of the genus are a source of diverse products for human needs, such as food, medicines, fibers, and beverages, and a good source of biomass for the production of biofuels, among many others. These plants are gaining importance as climate change becomes more evident as heat is reaching temperatures above 40 °C worldwide and rains are scarce. Many species of the genus grow in places where other plant species do not survive under severe field conditions, due to their CAM pathway for fixing CO2 where gas exchange occurs at night when stomata are open, allowing them to avoid excess loss of water. Most of the important species and varieties are usually propagated by offshoots that develop from rhizomes around the mother plant and by bulbils that develop up in the inflorescence, which are produced by the plant mostly when there is a failure in the production of seeds.Areas for commercial plantations are growing worldwide and therefore in the need of big amounts of healthy and good quality plantlets. Although many Agave species produce seeds, it takes longer for the plants to reach appropriate maturity and size for diverse purposes. Micropropagation techniques for the genus Agave offer the opportunity to produce relatively high amounts of plants year around in relatively small spaces in a laboratory. Here, a protocol for micropropagation that has proven good for several Agave species (including species from both subgenera) is presented in detail with two different kinds of explants to initiate the process: rescued zygotic embryos and small offshoots that grow around a mother plant.

Effect of prebiotics of Agave salmiana fed to healthy Wistar rats.

BACKGROUND: Inulin and other fructans are synthesized and stored in mezcal agave (Agave salmiana). Fructans provide several health benefits and have excellent technological properties, but only few data report their physiological effect when added in the diet. RESULTS: Here, we studied the physiological effects of fructans obtained from A. salmiana when added in the diet of Wistar rats. Results showed favorable changes on Wistar rats when the fructans was added to their diet, including the decrease of the pH in the feces and the increase of the number of lactic acid bacteria (CFU g-1 ) (Lactobacillus spp. and Bifidobacterium spp.), even these changes were enhanced with the synbiotic diet (fructans plus B. animalis subsp. lactis). Synbiotic diet, developed changes in the reduction of cholesterol and triglycerides concentrations in serum, with statistical differences (P < 0.05). Histological analysis of colon sections showed that synbiotic diet promoted colon cells growth suggesting that fructans from A. salmiana confer beneficial health effects through gut microbiota modulation. CONCLUSION: Our data underline the advantage of targeting the gut microbiota by colonic nutrients like specific structure of fructans from A. salmiana, with their beneficial effects. More studies are necessary to define the role of fructans to develop more solid therapeutic solutions in humans. © 2016 Society of Chemical Industry.

Effect of Agave americana and Agave salmiana Ripeness on Saponin Content from Aguamiel (Agave Sap).

Steroidal saponins have shown beneficial health effects. Agave spp. leaves and rhizomes are sources of these compounds, but their presence has not been reported in the aguamiel. Aguamiel is the sweet edible sap from mature agave, and its quality is influenced by the plant ripening stage. The purpose of this research was to identify and quantitate saponins in aguamiel from Agave americana and Agave salmiana at two ripening stages. Saponins and sapogenins were identified with HPLC/ESI-MS/TOF and quantitated with HPLC/ELSD. Results proved the presence of saponins derived from kammogenin, manogenin, gentrogenin, and hecogenin. The saponin content in aguamiel from immature A. salmiana was 2-fold higher (478.3 protodioscin equivalents (PE) µg/g aguamiel (DM)) compared with A. americana (179.0 PE µg/g aguamiel (DM)). In both species, saponin content decreased when plants reached sexual maturity. This should be considered before evaluating the effects of Agave spp. as a source of bioactive saponins.

Agave biotechnology: an overview.

Agaves are plants of importance both in Mexican culture and economy and in other Latin-American countries. Mexico is reported to be the place of Agave origin, where today, scientists are looking for different industrial applications without compromising its sustainability and preserving the environment. To make it possible, a deep knowledge of all aspects involved in production process, agro-ecological management and plant biochemistry and physiology is required. Agave biotechnology research has been focusing on bio-fuels, beverages, foods, fibers, saponins among others. In this review, we present the advances and challenges of Agave biotechnology.

Construction and evaluation of normalized cDNA libraries enriched with full-length sequences for rapid discovery of new genes from Sisal (Agave sisalana Perr.) different developmental stages.

To provide a resource of sisal-specific expressed sequence data and facilitate this powerful approach in new gene research, the preparation of normalized cDNA libraries enriched with full-length sequences is necessary. Four libraries were produced with RNA pooled from Agave sisalana multiple tissues to increase efficiency of normalization and maximize the number of independent genes by SMART™ method and the duplex-specific nuclease (DSN). This procedure kept the proportion of full-length cDNAs in the subtracted/normalized libraries and dramatically enhanced the discovery of new genes. Sequencing of 3875 cDNA clones of libraries revealed 3320 unigenes with an average insert length about 1.2 kb, indicating that the non-redundancy of libraries was about 85.7%. These unigene functions were predicted by comparing their sequences to functional domain databases and extensively annotated with Gene Ontology (GO) terms. Comparative analysis of sisal unigenes and other plant genomes revealed that four putative MADS-box genes and knotted-like homeobox (knox) gene were obtained from a total of 1162 full-length transcripts. Furthermore, real-time PCR showed that the characteristics of their transcripts mainly depended on the tight expression regulation of a number of genes during the leaf and flower development. Analysis of individual library sequence data indicated that the pooled-tissue approach was highly effective in discovering new genes and preparing libraries for efficient deep sequencing.

Fructan metabolism in A. tequilana Weber Blue variety along its developmental cycle in the field.

Fructan, as reserve carbohydrate, supplies energy needs during vegetative development, thereby exhibiting variations in its content and composition. Fructan metabolism in Agave tequilana Blue variety from 2- to 7-year-old plants was analyzed in this work. Soluble carbohydrates were determined at all ages. Fructan (328-711 mg/g), sucrose (14-39 mg/g), fructose (11-20 mg/g), glucose (4-14 mg/g), and starch (0.58-4.98 mg/g) were the most abundant carbohydrates. Thin-layer chromatography exhibited that 2-5-year-old plants mainly stored fructooligosaccharides, while 6-7-year-old plants mainly contained long-chain fructans. The fructan degree of polymerization (DP) increased from 6 to 23 throughout plant development. The 7-year-old plants mainly stored highly branched agavins. Partially methylated alditol acetate analyzed by gas chromatography-mass spectrometry reveals that fructan molecular structures became more complex with plant age. For the first time, we report the presence of a large number of DP3 (seven forms), DP4 (eight forms), and DP5 (six forms) isomers for agave fructans. Overall, fructan metabolism in A. tequilana displays changes in its soluble carbohydrates, DP, type, and fructan structures stored, along its developmental cycle in the field.

Polymorphism and methylation patterns in Agave tequilana Weber var. 'Azul' plants propagated asexually by three different methods.

Genetic variation in three forms of asexually propagated Agave tequilana Weber var. 'Azul' plants namely offsets, bulbils and in vitro cultured individuals was studied by AFLP analysis. Low levels of variation were observed between mother plants and offsets and a higher level between mother plant and bulbils. Families obtained from commercial plantations showed lower levels of variation in comparison to families grown as ornamentals. No variation was observed between the original explant and four generations of in vitro cultured plants. Epigenetic variation was also studied by analyzing changes in methylation patterns between mother plants and offspring in each form of asexual reproduction. Offsets and bulbils showed an overall decrease in methylation whereas in vitro cultured plants showed patterns specific to each generation: Generations 1 and 4 showed overall demethylation whereas Generations 2 and 3 showed increased methylation. Analysis of ESTs associated with transposable elements revealed higher proportions of ESTs from Ty1-copia-like, Gypsy and CACTA transposable elements in cDNA libraries obtained from pluripotent tissue suggesting a possible correlation between methylation patterns, expression of transposable element associated genes and somaclonal variation.

Agave tequilana MADS genes show novel expression patterns in meristems, developing bulbils and floral organs.

Agave tequilana is a monocarpic perennial species that flowers after 5-8 years of vegetative growth signaling the end of the plant's life cycle. When fertilization is unsuccessful, vegetative bulbils are induced on the umbels of the inflorescence near the bracteoles from newly formed meristems. Although the regulation of inflorescence and flower development has been described in detail for monocarpic annuals and polycarpic species, little is known at the molecular level for these processes in monocarpic perennials, and few studies have been carried out on bulbils. Histological samples revealed the early induction of umbel meristems soon after the initiation of the vegetative to inflorescence transition in A. tequilana. To identify candidate genes involved in the regulation of floral induction, a search for MADS-box transcription factor ESTs was conducted using an A. tequilana transcriptome database. Seven different MIKC MADS genes classified into 6 different types were identified based on previously characterized A. thaliana and O. sativa MADS genes and sequences from non-grass monocotyledons. Quantitative real-time PCR analysis of the seven candidate MADS genes in vegetative, inflorescence, bulbil and floral tissues uncovered novel patterns of expression for some of the genes in comparison with orthologous genes characterized in other species. In situ hybridization studies using two different genes showed expression in specific tissues of vegetative meristems and floral buds. Distinct MADS gene regulatory patterns in A. tequilana may be related to the specific reproductive strategies employed by this species.

Agave salmiana plant communities in central Mexico as affected by commercial use.

Agave salmiana is a native plant species harvested for the commercial production of mezcal (Agave spirits) in the highlands of central Mexico. The objective of this study was to identify vegetation changes in natural communities where A. salmiana has been differentially harvested for commercial purposes. Three plant community categories were identified in the state of Zacatecas based on their history of A. salmiana utilization: short (less than 10 years of use), moderate (about 25 years), and long (60 or more years). Species cover, composition, and density were evaluated in field surveys by use category. A gradient of vegetation structure of the communities parallels the duration of A. salmiana use. A. salmiana density was greatest (3,125 plants ha(-1)) in the short-use areas and less (892 plants ha(-1)) in the moderate-use areas, associated with markedly greater density of shrubs (200%) and Opuntia spp. (50%) in moderate-use areas. The main shrubs were Larrea tridentata, Mimosa biuncifera, Jatropha dioica and Buddleia scordioides while the main Opuntia species were Opuntia leucotricha and Opuntia robusta. A. salmiana density was least (652 plants ha(-1)) in the long-use areas where shrubs were less abundant but Opuntia spp. density was 25% higher than in moderate-use areas. We suggest that shrubs may increase with moderate use creating an intermediate successional stage that facilitates the establishment of Opuntia spp. Long-term Agave use is generating new plant communities dominated by Opuntia spp. (nopaleras) as a replacement of the original communities dominated by A. salmiana (magueyeras).

Germination, survival and growth of three vascular plants on biological soil crusts from a Mexican tropical desert.

Information about the effects of biological soil crusts (BSC) on germination, seedling survival and growth of vascular plants is controversial because they can have positive, neutral or negative effects. This controversy may be because most studies conducted until now have just analysed one or two recruitment stages independently. To understand the BSC effects on vascular plants, it is necessary to consider each stage of the recruitment process and synthesise all this information. The goal of this study was twofold. First, we analyse germination, seedling survival and growth of three vascular plants (Agave marmorata, Prosopis laevigata and Neobuxbaumia tetetzo) on BSC (cyanobacteria and mixed crust) from a tropical desert region of south-central México. Second, we synthesise the information to determine the total effect of BSC on plant species performance. We conducted experiments under controlled conditions to evaluate the proportion of germinated seeds, proportion of surviving seedlings and seedling dry weight in BSC and bare soil. Results showed that BSC have different effects on germination, seedling survival and growth of plant species. Plant species performance was qualitatively higher on BSC than bare soil. The highest performance of A. marmorata and P. laevigata was observed on cyanobacteria and mixed crusts, respectively. The highest performance of N. tetetzo was on both crust types.