Vascular anatomy
The largest 10% of petiole xylem vessels were significantly larger in diameter than the largest 10% of peduncle xylem vessels (P= 0.004) (fig. S4a). The mean diameter of the largest 10% of peduncle xylem vessels was 11.67μm, 30% smaller than the mean diameter of the largest 10% of petiole xylem vessels (16.81μm). Here, the largest 10% of vessels in both organs contributed the majority (52.70% ± 2.57%SE) of water volume flow through the tissues (fig. S4c). Although the vessel anatomy differed between the two organs, there was no significant difference (P=0.36) observed between the mean theoretical hydraulic conductivity of petioles and peduncles (fig S4b).
Discussion:
We demonstrate that fruit development (and thus by extension, seed production) in an annual plant species is prioritised at the expense of vegetative tissues under water stress through relatively greater xylem resistance to cavitation in those tissues supplying water to fruit. We found that leaf petiole and lamina xylem cavitated early during drought at a relatively high water potential, while the xylem of the main stem and peduncle remained functional until a lower water potential was reached. Under extreme water stress, tomato fruit continued growing, likely utilising water from leaf tissues released by cavitation, and by tissue capacitance observed through shrinkage. Phloem girdling showed that the xylem was the main pathway for water flow into the fruit during water stress. Thus, relatively cavitation resistant xylem vessels in the stem and peduncle provided a pathway supplying water to fruit from other tissues even when the plant was fully disconnected from a soil water source. The prioritization of reproductive tissues over vegetative tissues in drought is the opposite pattern to that observed in perennial species. (Fernandes et al., 2018; Bourbia et al., 2020).
Fruit is hydraulically prioritised through cavitation resistance.
The advantages of prioritising the water requirements of reproduction over vegetative tissues observed here in tomato become particularly apparent when considering the ancestry and evolution of this species. Tomato belongs to the Solanum section Lycopersicon in the Solanaceae that includes the cultivated tomato and 12 wild relatives (Peralta et al., 2007; Bergougnoux, 2014). While the specifics of tomato domestication are uncertain, the wild species originate in Western South America, growing from Northern Chile to Ecuador and the Galapagos Islands. These ancestral Lycopersicon occur in dry desert and pre-desert environments, including the Atacama Desert of Northern Chile where rainfall is highly sporadic, making the prioritisation of reproduction even under water stress a necessity for species persistence in the landscape (Chetelat et al., 2009; Fischer et al., 2011; Bergougnoux, 2014; Knapp & Peralta, 2016). In the case of opportunistic herbaceous species like tomato that tend to complete their life cycles during ephemeral rainfall, it appears advantageous under water stress to ensure all available water is directed to reproduction, thus safeguarding future offspring above the survival of the parent plant. We hypothesised that selection would favour individuals where xylem supply to fruiting tissue (in the stem and peduncle) was more resistant to cavitation than other organs, ensuring a water supply to fruit for as long as possible in drought conditions. The reasoning behind this prediction follows the hydraulic vulnerability segmentation hypothesis which considers the energetic or fitness cost of losing tissues to cavitation (Tyree & Ewers, 1991; Zimmermann, 2013; Johnson et al., 2016). Under the likely scenario that water becomes limited during reproduction (e.g. when growth was initiated after an ephemeral rain storm), the plant would be able to reproduce successfully, even if it required water to be extracted from the rest of the vegetative plant body. Given that stressors such as drought are known to induce flowering in many herbaceous species (Riboni et al., 2013; Riboni et al., 2016; Shavrukov et al., 2017) providing a so-called escape strategy from stress, we would expect hydraulic prioritization of reproduction to be an essential part of this ecological strategy.
Fruit water volume is supplied by xylem.
The idea of reproductive prioritisation by vulnerability segmentation depends on the assumption that the water reaching fruit is supplied by xylem and thus exposed to the tension and cavitation risk experienced by the broader plant in drought. The proportion of water proposed to be supplied to the tomato fruit by the xylem ranges from 10 to 90% (Ho et al., 1987; Windt et al., 2009). However, we found that phloem girdled fruit continued growing at the same rate as ungirdled fruit under water stress indicating that in this species, at least during water stress, fruit hydration is predominantly supplied by the xylem. This result is supported by previous studies in tomato (Windt et al., 2009; Van De Wal et al., 2017), although this xylem connection may deteriorate during later fruit development (Li et al., 2021). By demonstrating that the xylem is the main source of water supplying reproductive tissues even during periods of water stress, these data emphasize the special relevance of the cavitation resistance of xylem in the peduncle for understanding fruit growth and survival in water stress. In this case, identifying xylem cavitation characteristics in reproductive tissues is highly relevant to understanding the ecology and reproductive behaviour of plant species.
We anticipated that the capacitance from shrinking tissue or early cavitation of vulnerable organs would make water stored in these tissues available to more resistant tissues, provided a xylem connection remained intact between them (Cochard & Tyree, 1990; Hölttä et al., 2009; Johnson et al., 2016; Bourbia et al., 2020). Given the relative vulnerability of leaves compared with reproductive tissues, we argue that the collapse and early cavitation of non-prioritised leaves liberates water from these tissues (Hölttä et al., 2009), while a strong xylem connection remains intact under powerful dehydration stress allowing an intact passage of water from the vulnerable leaves to the hydraulically prioritised fruit. Different xylem anatomies may play a role in facilitating the variation in xylem vulnerability between reproductive and vegetative organs. Here, the small vessels of peduncles appeared more resistant to cavitation than petioles, supporting literature indicating a relationship between vessel size and cavitation vulnerability (Cochard & Tyree, 1990; Hacke et al., 2001; Jacobsen et al., 2005; Petit et al., 2009; Lens et al., 2011; Scoffoni et al., 2017; Gauthey et al., 2020), however this idea has been contested (Rodriguez-Dominguez et al., 2018). Although not examined here, xylem pit anatomy has been strongly linked to xylem vulnerability (Lens et al., 2011; Zhang et al., 2020), and may provide a mechanistic explanation for the observed vulnerability segmentation observed between organs (Thonglim et al., 2021). Although these organs differed significantly in xylem vessel size distribution and P50, the theoretical hydraulic conductance of the organs did not differ significantly. This is interesting considering the differences in photosynthetic and reproductive water requirements of leaves and fruit and reinforces the importance of xylem supply to fruiting tissues.
Reproductive hydraulic vulnerability: a life history trait.
Fruit prioritisation in this herbaceous annual differs significantly to the observations in woody and herbaceous species with a perennial life history. Evidence in Pyrethrum, a perennial daisy, suggests that the isolation of leaky flowers by early cavitation may delay dehydration damage to the vegetative tissue of plants (Bourbia et al., 2020). In the perennial case, the early cavitation of leaky petals and peduncles during drought supports the survival of vegetative tissues (Bourbia et al., 2020). This perennial strategy would allow the vegetative plant to persist through drought, enabling reproduction under more favourable future conditions. The susceptibility of fruit to water limitation has also been shown in woody perennial species such as olive (Fernandes et al., 2018). These divergent patterns in annual and perennial species suggest strong hydraulic selection based on life history, with reproduction heavily prioritised in the annual plant, and readily sacrificed in perennial species. For annual plants in conditions where water is limiting, it appears advantageous to prioritise successful reproduction over the preservation of leaf tissues. Alternately, for perennial plants, reproduction under drought conditions represents a water cost that can be deferred.
Like the perennial daisy, tomato may have mechanisms to isolate leaky flowers and fruit in immature developmental stages (Reichardt et al., 2020). This is suggested by our observation that the tomato petals showed no greater cavitation resistance than leaf tissues. Given these findings, established tomato fruit appear heavily hydraulically prioritised, while immature (and less reproductively viable) structures like flowers are rapidly sacrificed, eliminating potentially large floral evaporative water costs to the plant (Feild et al., 2009b; Teixido & Valladares, 2014; Roddy et al., 2018; Bourbia et al., 2020). This suggests a transition point at which established fruit with a high chance of successful seed production receive greater hydraulic investment.
Conclusions:
Including hydraulic vulnerability segmentation in the suite of life history traits that characterise annual and perennial plants provides a novel perspective on the variation in their ecological strategies. However, the xylem vulnerability of whole plants in drought, including their reproductive tissues, has yet to be fully examined across broader plant diversity. If these divergent patterns of reproductive vulnerability can be demonstrated to be typical of annual and perennial plants, then these hydraulic traits may significantly contribute to our understanding of plant behaviour in changing climates based on life history. In the annual species tomato we see fruit prioritisation driven through whole plant hydraulic relationships, ensuring fruit survival and seed production through greater cavitation resistance in reproductive tissues relative to leaves. Our findings highlight the need to include reproductive tissues when examining whole plant water relations. These results are likely to be relevant to other crop species, particularly if prioritisation of reproduction through xylem resistance can be shown to be a common annual species trait. As temperatures and drought events increase globally, understanding the plant water transport traits that limit reproductive performance has direct applications for both crop production and conservation. Our results indicate xylem resistance to cavitation in the support structures of reproductive tissues is key to the success of fruit in water stress, a trait with potential implications for breeding resilient crops and climate provenanced species.
Author Contribution:
B.L.H.D., M.R.C-M. and T.J.B designed the experiment, B.L.H.D performed the experiment with assistance from M.R.C-M. and T.J.B. B.L.H.D. processed and interpreted the data with assistance from M.R.C-M. and T.J.B. B.L.H.D wrote the manuscript, and all authors reviewed and commented on the manuscript.
Data availability statement: The data that support the findings of this study are available from the corresponding author upon reasonable request.
References:
Adams HD, Zeppel MJB, Anderegg WRL, Hartmann H, Landhäusser SM, Tissue DT, Huxman TE, Hudson PJ, Franz TE, Allen CD, et al. 2017. A multi-species synthesis of physiological mechanisms in drought-induced tree mortality. Nature Ecology & Evolution 1(9): 1285-1291.
Anderegg WR, Flint A, Huang C-y, Flint L, Berry JA, Davis FW, Sperry JS, Field CB. 2015. Tree mortality predicted from drought-induced vascular damage. Nature Geoscience 8(5): 367-371.
Anderegg WR, Martinez-Vilalta J, Cailleret M, Camarero JJ, Ewers BE, Galbraith D, Gessler A, Grote R, Huang C-Y, Levick SR, et al. 2016. When a Tree Dies in the Forest: Scaling Climate-Driven Tree Mortality to Ecosystem Water and Carbon Fluxes. Ecosystems 19(6): 1133-1147.
Bergougnoux V. 2014. The history of tomato: From domestication to biopharming. Biotechnology Advances 32(1): 170-189.
Bertin N. 2003. A Model for an Early Stage of Tomato Fruit Development: Cell Multiplication and Cessation of the Cell Proliferative Activity. Annals of botany 92(1): 65-72.
Bourbia I, Carins‐Murphy MR, Gracie A, Brodribb TJ. 2020. Xylem cavitation isolates leaky flowers during water stress in pyrethrum. New Phytologist 227(1): 146-155.
Brodribb TJ, Bienaimé D, Marmottant P. 2016a. Revealing catastrophic failure of leaf networks under stress. Proceedings of the National Academy of Sciences 113(17): 4865-4869.
Brodribb TJ, Carriqui M, Delzon S, Lucani C. 2017. Optical Measurement of Stem Xylem Vulnerability. Plant Physiology 174(4): 2054-2061.
Brodribb TJ, Powers J, Cochard H, Choat B. 2020. Hanging by a thread? Forests and drought. Science 368(6488): 261-266.
Brodribb TJ, Skelton RP, McAdam SA, Bienaime D, Lucani CJ, Marmottant P. 2016b. Visual quantification of embolism reveals leaf vulnerability to hydraulic failure. New Phytol 209(4): 1403-1409.
Bussières P. 1994. Water Import Rate in Tomato Fruit: A Resistance Model. 73(1): 75-82.
Chapotin SM, Holbrook NM, Morse SR, Gutierrez MV. 2003. Water relations of tropical dry forest flowers: pathways for water entry and the role of extracellular polysaccharides. Plant Cell and Environment 26(4): 623-630.
Chetelat RT, Pertuzé RA, Faúndez L, Graham EB, Jones CM. 2009. Distribution, ecology and reproductive biology of wild tomatoes and related nightshades from the Atacama Desert region of northern Chile. Euphytica 167(1): 77-93.
Choat B, Brodribb TJ, Brodersen CR, Duursma RA, López R, Medlyn BE. 2018. Triggers of tree mortality under drought. Nature 558(7711): 531-539.
Choat B, Jansen S, Brodribb TJ, Cochard H, Delzon S, Bhaskar R, Bucci SJ, Feild TS, Gleason SM, Hacke UG, et al. 2012. Global convergence in the vulnerability of forests to drought. Nature 491(7426): 752-755.
Cochard H, Tyree MT. 1990. Xylem dysfunction in Quercus: vessel sizes, tyloses, cavitation and seasonal changes in embolism. Tree Physiology 6(4): 393-407.
De La Barrera E, Nobel PS. 2004. Nectar: properties, floral aspects, and speculations on origin. Trends in Plant Science 9(2): 65-69.
Feild T, Chatelet DS, Brodribb TJ. 2009a. Ancestral xerophobia: a hypothesis on the whole plant ecophysiology of early angiosperms. Geobiology 7(2): 237-264.
Feild T, Chatelet DS, Brodribb TJ. 2009b. Giant Flowers of Southern Magnolia Are Hydrated by the Xylem. Plant Physiology 150(3): 1587-1597.
Fernandes RDM, Cuevas MV, Diaz-Espejo A, Hernandez-Santana V. 2018. Effects of water stress on fruit growth and water relations between fruits and leaves in a hedgerow olive orchard. Agricultural water management 210: 32-40.
Fischer I, Camus-Kulandaivelu L, Allal F, Stephan W. 2011. Adaptation to drought in two wild tomato species: the evolution of the Asr gene family. New Phytologist 190(4): 1032-1044.
Galen C, Sherry RA, Carroll AB. 1999. Are flowers physiological sinks or faucets? Costs and correlates of water use by flowers of Polemonium viscosum. Oecologia 118(4): 461-470.
Gauthey A, Peters JMR, Carins‐Murphy MR, Rodriguez‐Dominguez CM, Li X, Delzon S, King A, López R, Medlyn BE, Tissue DT, et al. 2020. Visual and hydraulic techniques produce similar estimates of cavitation resistance in woody species. New Phytologist.
Gillaspy G, Ben-David H, Gruissem W. 1993. Fruits: a developmental perspective. The Plant Cell 5(10): 1439.
Hacke UG, Sperry JS, Pockman WT, Davis SD, McCulloh KA. 2001. Trends in wood density and structure are linked to prevention of xylem implosion by negative pressure. Oecologia 126(4): 457-461.
Hanssens J, De Swaef T, Steppe K. 2015. High light decreases xylem contribution to fruit growth in tomato. Plant, Cell & Environment 38(3): 487-498.
Hedhly A, Hormaza JI, Herrero M. 2009. Global warming and sexual plant reproduction. Trends in Plant Science 14(1): 30-36.
Higuchi H, Sakuratani T. 2005. The Sap Flow in the Peduncle of the Mango (Mangifera indica L.) Inflorescence as Measured by the Stem Heat Balance Method. Journal of the Japanese Society for Horticultural Science 74(2): 109-114.
Higuchi H, Sakuratani T. 2006. Water Dynamics in Mango (Mangifera indica L.) Fruit during the Young and Mature Fruit Seasons as Measured by the Stem Heat Balance Method. Journal of the Japanese Society for Horticultural Science 75(1): 11-19.
Ho L. 1980. Control of import into tomato fruits. Berichte der Deutschen Botanischen Gesellschaft 93(1): 315-325.
Ho L, Grange RI, Picken AJ. 1987. An analysis of the accumulation of water and dry matter in tomato fruit. Plant, Cell and Environment 10(2): 157-162.
Ho L, Sjut V, Hoad G. 1982. The effect of assimilate supply on fruit growth and hormone levels in tomato plants. Plant Growth Regulation 1(3): 155-171.
Hölttä T, Cochard H, Nikinmaa E, Mencuccini M. 2009. Capacitive effect of cavitation in xylem conduits: results from a dynamic model. Plant, Cell & Environment 32(1): 10-21.
Jacobsen AL, Ewers FW, Pratt RB, Paddock WA, Davis SD. 2005. Do Xylem Fibers Affect Vessel Cavitation Resistance? Plant Physiology 139(1): 546-556.
Johnson DM, Wortemann R, Mcculloh KA, Jordan-Meille L, Ward E, Warren JM, Palmroth S, Domec J-C. 2016. A test of the hydraulic vulnerability segmentation hypothesis in angiosperm and conifer tree species. Tree Physiology 36(8): 983-993.
Knapp S, Peralta IE 2016. The Tomato (Solanum lycopersicum L., Solanaceae) and Its Botanical Relatives. Compendium of Plant Genomes: Springer Berlin Heidelberg, 7-21.
Lambrecht SC. 2013. Floral Water Costs and Size Variation in the Highly Selfing (Polemoniaceae). International journal of plant sciences 174(1): 74-84.
Lens F, Sperry JS, Christman MA, Choat B, Rabaey D, Jansen S. 2011. Testing hypotheses that link wood anatomy to cavitation resistance and hydraulic conductivity in the genus Acer. New Phytologist 190(3): 709-723.
Lesk C, Rowhani P, Ramankutty N. 2016. Influence of extreme weather disasters on global crop production. Nature 529(7584): 84-87.
Li H, Zhang X, Hou X, Du T. 2021. Developmental and water deficit-induced changes in hydraulic properties and xylem anatomy of tomato fruit and pedicel. Journal of Experimental Botany.
Matthews MA, Shackel KA 2005. Growth and water transport in fleshy fruit. Vascular transport in plants: Elsevier, 181-197.
Peralta I, Knapp S, Spooner D. 2007. The taxonomy of tomatoes: a revision of wild tomatoes (Solanum L. section Lycopersicon (Mill.) Wettst.) and their outgroup relatives (Solanum sections Juglandifolium (Rydb.) Child and Lycopersicoides (Child) Peralta). Systematic Botany Monographs 84: 1-186.
Petit G, Anfodillo T, De Zan C. 2009. Degree of tapering of xylem conduits in stems and roots of small Pinus cembra and Larix decidua trees. Botany 87(5): 501-508.
Prasad P, Staggenborg S, Ristic Z. 2008. Impacts of drought and/or heat stress on physiological, developmental, growth, and yield processes of crop plants. Response of crops to limited water: Understanding and modeling water stress effects on plant growth processes(responseofcrops): 301-355.
Reichardt S, Piepho H-P, Stintzi A, Schaller A. 2020. Peptide signaling for drought-induced tomato flower drop. Science 367(6485): 1482-1485.
Renaudin J-P, Deluche C, Cheniclet C, Chevalier C, Frangne N. 2017. Cell layer-specific patterns of cell division and cell expansion during fruit set and fruit growth in tomato pericarp. Journal of Experimental Botany 68(7): 1613-1623.
Riboni M, Galbiati M, Tonelli C, Conti L. 2013. GIGANTEA Enables Drought Escape Response via Abscisic Acid-Dependent Activation of the Florigens and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1. Plant Physiology 162(3): 1706-1719.
Riboni M, Robustelli Test A, Galbiati M, Tonelli C, Conti L. 2016. ABA-dependent control ofGIGANTEAsignalling enables drought escape via up-regulation ofFLOWERING LOCUS TinArabidopsis thaliana. Journal of Experimental Botany 67(22): 6309-6322.
Roddy AB. 2019. Energy Balance Implications of Floral Traits Involved in Pollinator Attraction and Water Balance. International journal of plant sciences 180(9): 944-953.
Roddy AB, Jiang GF, Cao K, Simonin KA, Brodersen CR. 2019. Hydraulic traits are more diverse in flowers than in leaves. New Phytologist 223(1): 193-203.
Roddy AB, Simonin KA, Mcculloh KA, Brodersen CR, Dawson TE. 2018. Water relations of Calycanthus flowers: Hydraulic conductance, capacitance, and embolism resistance. Plant, Cell & Environment 41(10): 2250-2262.
Rodriguez-Dominguez CM, Carins Murphy MR, Lucani C, Brodribb TJ. 2018. Mapping xylem failure in disparate organs of whole plants reveals extreme resistance in olive roots. New Phytologist 218(3): 1025-1035.
Savi T, Bertuzzi S, Branca S, Tretiach M, Nardini A. 2015. Drought-induced xylem cavitation and hydraulic deterioration: risk factors for urban trees under climate change? New Phytologist 205(3): 1106-1116.
Scoffoni C, Albuquerque C, Brodersen CR, Townes SV, John GP, Cochard H, Buckley TN, McElrone AJ, Sack L. 2017. Leaf vein xylem conduit diameter influences susceptibility to embolism and hydraulic decline. New Phytologist 213(3): 1076-1092.
Shameer S, Vallarino JG, Fernie AR, Ratcliffe RG, Sweetlove LJ. 2020. Flux balance analysis of metabolism during growth by osmotic cell expansion and its application to tomato fruits. Plant Journal 103(1).
Shavrukov Y, Kurishbayev A, Jatayev S, Shvidchenko V, Zotova L, Koekemoer F, de Groot S, Soole K, Langridge P. 2017. Early flowering as a drought escape mechanism in plants: How can it aid wheat production? Frontiers in plant science 8: 1950.
Skelton RP, Brodribb TJ, Choat B. 2017. Casting light on xylem vulnerability in an herbaceous species reveals a lack of segmentation. New Phytologist 214(2): 561-569.
Soltis PS, Soltis DE. 2014. Flower diversity and angiosperm diversification. Methods Mol Biol 1110: 85-102.
Sperry JS, Love DM. 2015. What plant hydraulics can tell us about responses to climate-change droughts. New Phytologist 207(1): 14-27.
Sperry JS, Tyree MT. 1988. Mechanism of Water Stress-Induced Xylem Embolism. Plant Physiology 88(3): 581-587.
Teixido AL, Valladares F. 2014. Disproportionate carbon and water maintenance costs of large corollas in hot Mediterranean ecosystems. Perspectives in Plant Ecology, Evolution and Systematics 16(2): 83-92.
Thonglim A, Delzon S, Larter M, Karami O, Rahimi A, Offringa R, Keurentjes JJ, Balazadeh S, Smets E, Lens F. 2021. Intervessel pit membrane thickness best explains variation in embolism resistance amongst stems of Arabidopsis thaliana accessions. Annals of botany 128(2): 171-182.
Tyree MT, Ewers FW. 1991. The hydraulic architecture of trees and other woody plants. New Phytologist 119(3): 345-360.
Tyree MT, Sperry JS. 1989. Vulnerability of xylem to cavitation and embolism. Annual review of plant biology 40(1): 19-36.
Urli M, Porté AJ, Cochard H, Guengant Y, Burlett R, Delzon S. 2013. Xylem embolism threshold for catastrophic hydraulic failure in angiosperm trees. Tree Physiology 33(7): 672-683.
Van De Wal BAE, Windt CW, Leroux O, Steppe K. 2017. Heat girdling does not affect xylem integrity: anin vivomagnetic resonance imaging study in the tomato peduncle. New Phytologist 215(2): 558-568.
van Die J, Willemse NC. 1980. The supply of water and solutes by phloem and xylem to growing fruits of Yucca flaccida Haw. Berichte der Deutschen Botanischen Gesellschaft 93(1): 327-337.
Van Ieperen W, Volkov VS, Van Meeteren U. 2003. Distribution of xylem hydraulic resistance in fruiting truss of tomato influenced by water stress. Journal of Experimental Botany 54(381): 317-324.
Vilagrosa A, Chirino E, Peguero-Pina JJ, Barigah TS, Cochard H, Gil-Pelegrín E 2012. Xylem Cavitation and Embolism in Plants Living in Water-Limited Ecosystems. Plant Responses to Drought Stress: Springer Berlin Heidelberg, 63-109.
Windt CW, Gerkema E, Van As H. 2009. Most Water in the Tomato Truss Is Imported through the Xylem, Not the Phloem: A Nuclear Magnetic Resonance Flow Imaging Study. Plant Physiology 151(2): 830-842.
Zhang F-P, Brodribb TJ. 2017. Are flowers vulnerable to xylem cavitation during drought? Proceedings of the Royal Society B: Biological Sciences 284(1854): 20162642.
Zhang F-P, Carins Murphy MR, Cardoso AA, Jordan GJ, Brodribb TJ. 2018. Similar geometric rules govern the distribution of veins and stomata in petals, sepals and leaves. New Phytologist 219(4): 1224-1234.
Zhang F-P, Zhang J-L, Brodribb TJ, Hu H. 2020. Cavitation resistance of peduncle, petiole and stem is correlated with bordered pit dimensions in Magnolia grandiflora. Plant Diversity.
Zimmermann MH. 2013. Xylem structure and the ascent of sap: Springer Science & Business Media.