Morpho-physiological traits associated with contrasting water-use efficiency in Piper nigrum

Morpho-physiological traits associated with contrasting water-use efficiency in Piper nigrum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Morpho-physiological traits associated with contrasting water-use efficiency in Piper nigrum Helane CA Santos, Joaquim AL Junior, Olavo P Silva, Rafaela S Guerino, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4412806/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Water-use efficiency (WUE) also known as crop-per-drop has been the focus of several studies concerning the limitation of water and natural resources. Alongside this, morpho-physiological aspects underlying WUE in many species have been exploited to be set up to different water regimes. Here, two cultivars of Piper nigrum (Clonada and Uthirankotta), growing under an irrigation system, were investigated for morpho-physiological aspects linked to WUE by accessing anatomical, morphological, photosynthetic, and hydraulic parameters. Our findings reveal that cv. Uthirankotta presents a higher water-use efficiency at the whole-plant level (WUE yield ) than cv. Clonada. However, despite this difference, no association between short-term water-use efficiency (WUE E and WUE gs ) and long-term water-use efficiency (WUE yield ) was observed for both cultivars. Such responses were instead linked to divergence in structural and functional traits observed in growth, anatomy, and hydraulic parameters between such plant materials. We believe that our report can support further studies addressing WUE in Piper nigrum under contrasting water availability by assessing underlying parameters closely associated with long- rather than short-term WUE. black pepper gas exchange water status plant hydraulic conductance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key message Distinct morpho-physiological traits rather than gas exchange at leaf levels underpin water-use efficiency in Piper nigrum Headings Morpho-physiological traits related to WUE in Piper nigrum Introduction Originally from tropical regions of India, the black pepper ( Piper nigrum ) is a perennial, semi-woody, shrubby, and climbing plant, that grows attached to wooden stakes or tree trunks (Lima et al. 2010; Ahmad et al. 2012; Rasanjali et al. 2019). This species holds stems formed by two distinct parts: the central stem, which presents adventitious and staple-like roots (orthotropic branch), and the lateral stems, which are devoid of adherent roots and where buds sprout into flowers and fruits, thus referred to as plagiotropic branches (EMBRAPA, 2004; Serrano et al. 2012; Martins 2018). As an understory plant, Piper nigrum is adapted to dappled light and temperatures between 23 and 28 °C, with relative air humidity ranging from 80 to 90%, thereby requiring a hot and humid climate, with total annual rainfall above 1,500 mm during the flowering and fruiting set period (Duarte et al. 2006; Andrade, Silva, Salles 2017). Over the recent decades, P. nigrum has been cultivated in fields under full sun and this transition has impacted overall fit and yield (EMBRAPA 2004; Oliveira et al. 2018; Sulistyaningsih et al. 2021). Leaf morpho-anatomical changes comprise one of the several strategies used by plants to acclimate to different edaphoclimatic conditions (Cal et al. 2019; Ding et al. 2020). The success of such modifications is strictly associated with growth through improvements in carbon dioxide (CO 2 ) assimilation and water loss control (Mitchell et al. 2013). In this context, it is well-known that leaf morpho-anatomical changes play a crucial role in influencing photosynthesis and regulating water loss, and these modifications primarily impact resistance to gas movement (Mitchell et al. 2013; Ding et al. 2020). Among the known leaf gas flow resistances, stomatal, mesophilic, and cuticular are highlighted (Xiao et al. 2017; Guzmán-Delgado et al. 2021). Stomatal resistance refers to the resistance to gas diffusion through the stomata, while mesophyll resistance refers to the impedance to gas diffusion between the cells of the chlorophyll parenchyma, and both are directly related to anatomical characteristics of the leaf (Ding et al. 2020; Guzmán‐Delgado et al. 2021). Moreover, it has been demonstrated that leaf morphology also responds to the transition from shadow to sun in several plants (Yang et al. 2013; Rodríguez-Lopez et al. 2014; Sevillano et al. 2016). In this context, plants adapted to the sun typically have thicker leaves coupled to developed vascular systems compared to shade plants (Kim et al. 2005; Mathur et al. 2018). On the other hand, shade plants may have thinner leaves with less pronounced vascular structures, as they typically experience lower rates of transpiration and need to maximize light capture efficiency (Fritz et al. 2018; Mathur et al. 2018). These adaptations reflect how plants optimize their leaf morphology and physiology to efficiently use available light and water resources (Rodríguez-Lopez et al. 2014; Sevillano et al. 2016); however, how these features impact fit and yield in P. nigrum are still elusive. Water-use efficiency (WUE) in plants is claimed as the ability of a plant to effectively use water for growth and physiological processes while minimizing water loss. It is a key determinant of plant productivity, particularly in regions with limited water availability or during periods of drought stress (Briggs and Shantz 1913; Bramley et al. 2013; Flexas et al. 2013). WUE can be estimated in many ways, which spans either organ to whole-plant levels or short to long-term responses (Hatfield and Dold 2019; Petrík et al. 2023). In general, divergent data have been raised in the literature when WUE at the leaf (WUE E , WUE gs ) and the whole-plant levels (WUE plant ) are compared (Blum 2008; Tomás et al. 2012; Medrano et al. 2015). This response is not immediately understood but has been hypothesized to be involved not only with gas exchange parameters but also with other parameters such as morphological and hydraulic traits (Yang et al. 2013; Medrano et al. 2015; Hatfield and Dold 2019). In this scenario, P. nigrum has shown a pronounced sensitivity to drought and exhibits varied responses in WUE under such conditions (Ambrozim et al. 2022; Teles et al. 2023). However, the structural and functional aspects associated with cultivars showing distinct WUE remain to be thoroughly examined. Improving water-use efficiency in plants is essential for sustainable agriculture, food security, and ecosystem resilience, particularly in a scenario of climate change and water scarcity (Bertolino et al. 2019; Petrík et al. 2023). Extensive efforts have been made to decipher mechanisms underlying WUE and develop strategies to improve it in both agricultural and natural systems (Gago et al. 2014; Bertolino et al. 2019; Hatfield and Dold 2019). In this sense, WUE is a determining factor for the set up and management of irrigation systems, thus reducing water loss (Yang et al 2015; Linker et al. 2016; Kang et al. 2021). However, despite the association between morpho-physiological aspects and WUE has been documented elsewhere (Dittberner et al. 2018; Bertolino et al. 2019; Ma et al. 2023; Petrík et al. 2023), there is a lack of evidence for P. nigrum . Herein, morpho-physiological aspects underlying water-use efficiency were investigated by assessing growth, anatomy, gas exchange, and hydraulic parameters in two cultivars of Piper nigrum . Moreover, aspects related to the association between water-use efficiency at leaf and whole-plant levels are also discussed. Materials and Methods Plant material and experimental conditions The experiment was conducted at fields of the Tropical Products Company of Castanhal (Tropoc), located in the municipality of Castanhal, PA, Brazil (01º 17' 50” S, 47º 55' 20” W, 40 m altitude). The study was carried out in partnership with the Study Group on Water and Soil Engineering in the Amazon (GEEASA) from the Universidade Federal Rural da Amazônia (UFRA). The field is composed of 2-year-old seedlings from two cultivars of Piper nigrum L., namely cv. Clonada and cv. Uthirankotta. They were arranged in an area of 1930 m 2 . The experiment is set up in a randomized blocks design, in which each plot consisted of four plants per cultivar (Clonada and Uthirankotta), in a double row, with a spacing of 4.0 m between rows and 2.20 x 2.20 m among plants. The cv. Clonada was obtained from seed germination and in vitro of the Kuthiravally cultivar, an early fruit maturation material, which normally sprouts from December to February, with a period of pollination to fruit maturation of six months. It displays medium-sized foliage, purpure-colored sprouts, long ears with good filling, and medium-sized fruits, with an average production of 3 kg plant -1 (Fig. S1). On the other hand, cv. Uthirankotta is a well-established cultivar in the region. It holds a late fruit maturation cycle, normally the flowering from January to March, with a phase of six months from pollination to fruit maturation. It presents wide leaves, long ears containing several rows of fruits, dark-purple-colored sprouts, and medium-sized fruits, with an average production of 3 kg plant -1 (Fig. S1). The soil classification is a dystrophic Yellow Argisol (medium texture), with a predominance of secondary vegetation (Cardoso Júnior et al. 2007). The soil chemical and acidity corrections were carried out by applying liming (3.7 t ha -1 ) and both organic and mineral planting fertilizers as previously described by Oliveira and Nakayama (2007). Plants were cultivated under global radiation (GR) of 1303 W/m 2 (nearly 700 µmol de photons m -2 s -1 ), and daytime temperatures of 25.3 to 26.2 ± 2 °C (day/night), with relative air humidity between 60.5 and 86%. A total precipitation of 1,520 mm, with rainfall occurring in January (489 mm) and May (388 mm) and the lowest in February (99.8 mm) (Fig. S2). To confirm that all plants were kept at field capacity over the course of this experiment, the soil moisture was monitored with tensiometers installed at a depth of 20 cm-30cm. The tensiometers were positioned in line with the culture, 15 cm from the drippers. Readings were taken daily at 8:30 am. The analyses of gas exchange and water status were performed 24 months after the experiment was initiated. The yield was determined at the end of the harvest season. The entire experiment period comprised 36 months. Yield and water-use efficiency Fruits were harvested during their respective seasons for each cultivar (between July and October for cv. Clonada, and August to September for cv. Uthirankotta). Based on pepper fruits fresh weight, the yield (Y) was calculated as Y=P/A, where Y, as yield (kg ha -1 ); P, as production (kg); and A, as area (ha). The water balance over the experiment period was monitored and expressed in mm. Thus, the water-use efficiency (WUE yield ) was accomplished through the relationship between black pepper fruit productivity (kg ha -1 ) and water consumption (mm) (Doorenbos and Kassam 1994), as WUE yield =Y/w, where: WUE yield , as water-use efficiency, kg ha -1 mm -1 ; Y, as total yield, kg ha -1 ; w, as volume of water applied, mm. Gas exchange Gas exchange parameters were determined using the infrared gas analyzer (LCpro-SD, ADC BioScientific Ltd, United Kingdom). Analyzes were performed on the third to fourth fully expanded leaf of the twelfth branch from the base. The analyses included net carbon assimilation rate ( A ), stomatal conductance ( g s ), transpiration ( E ), and intercellular CO 2 concentration ( C i ), which were measured under photosynthetically active radiation (PAR) of 1000 µmol (photons) m –2 s –1 and 400 ppm CO 2 at leaf level (Yin et al. 2009). Instantaneous (WUE E ) and intrinsic water-use efficiency (WUE gs ) were estimated based on the ratio between A / E and A / g s , respectively. Response curves of net photosynthesis ( A ) to photosynthetically active radiation (PAR) were also carried out. For this, leaves were submitted to 12 points of light intensity, varying the photosynthetically active radiation from 0 to 1400 µmol photons m -2 s -1 via a light source (halogen bulb with 50W reflector) under a fixed concentration of 400 µmol mol -1 air (C a ) of CO 2 in the chamber. The photosynthetic light-response curve ( A/ PAR curve) were adjusted as described by Lobo et al. (2013). From the A /PAR curve plots, light compensation point ( I c ), light saturation point ( I s ), photosynthesis rate in saturating light ( A sat ), and maximum gross photosynthesis rate ( A max ) were calculated by fitting the mechanistic model (Lobo et al. 2013). Water status Leaf water potential measurements were carried out at both predawn (Ψ pd ) and midday (Ψ md ) using the Scholander pressure chamber (Scholander et al. 1965), model M 1505D (Pressure Chamber Instruments, PMS). The Ψ pd was performed between 2:30 am and 5:30 am, while the Ψ md was carried out between 12:00 pm and 3:00 pm. Afterwards, the plant's hydraulic conductance ( K plant ) was estimated as described by Kramer and Boyer (1995), and Avila et al. (2020), in which K plant = E /([-(Ψ md – Ψ pd )]); where K plant, as hydraulic conductance of the plant (mmol H 2 O m -2 s -1 MPa -1 ); E , plant leaf transpiration (mmol H 2 O m -2 s -1 ); Ψ md , midday water potential (MPa); Ψ pd , predawn water potential (MPa). Parameters based on the pressure-volume (PV) curve (Ψ w x RWC) were determined on the third to fourth fully expanded leaf of the twelfth branch from the base. Two leaves were collected per plot and block, totaling 6 leaves per cultivar. Leaves were cut under water and rehydrated overnight until Ψ w reached nearly −0.1 MPa. Both leaf weight and Ψ w were recorded using the Scholander pressure chamber and precision scale (de 0,001 g) over time during desiccation on the laboratory bench until Ψ w stabilization. At least four to five points were collected before and after the turgor loss point for each leaf (Tyree and Hammel 1972). Except for the first weighing (considered as FW 1 ) of each leaf per cultivar, the others were considered as fresh weight (FW 2 ). The leaves were dried in an oven at 65 ºC, and the dry mass (DW) was recorded. Then, the relative water content (RWC) at each point was calculated as RWC (%) = ­(FW 1 − DW)∕FW 2 − DW) × 100. Finally, the osmotic potential at full turgor (Ψ s100% ), osmotic potential at turgor loss point (Ψ sTLP ), bulk elastic module (Ꜫ v ), relative water content at field capacity (RWC 100% ) and relative water content at turgor loss point (RWC TLP ) were estimated based on recommendations of Cardoso et al. (2018) and Cardoso et al. (2020). Growth Parameters Total leaf area (TLA) was determined as described by Rhoads and Bloodworth (1964). For this, leaves were collected in the portion corresponding to ¼ of the plant height. The leaves were placed in paper bags and transferred to an oven at 65º C for 72 h. Afterwards, the leaves were weighed on an analytical scale and then TLA was estimated according to Reddy et al. (1989): TLA=FW leaf x SLA x 4. TLA, total average leaf area of a plant (m 2 ); FW leaf , dry weight of leaves corresponding to ¼ of the plant height (kg); SLA, specific leaf area (g m 2 ); 4, as a constant term. The specific leaf area (SLA) was determined as described by Hunt (1982). Morpho-anatomical analyses Morpho-anatomical analyses were carried out in the median region of the fourth fully expanded leaf of the twelfth branch (from base to apex) in 3-year-old plants. Samples were immediately fixed in FAA70 (70% formaldehyde-acetic acid-ethanol) in a ratio of 1:1:18 (Johansen 1940) for 24 h and subsequently stored in 70% alcohol. This material was embedded in methacrylate (Historesin-Leica) following the manufacturer's recommendations. Subsequently, samples were transversely sectioned (5 μm thick) with an automatic feeding rotary microtome (model RM2155, Leica Microsystems Inc., Heidelberg, Germany) and stained with toluidine blue (O'Brien et al. 1964). To assess leaf thickness (LT) mesophyll thickness (MT), adaxial epidermis thickness (AdET), and abaxial epidermis thickness (AbET), the fragments of the median region of the leaves were immersed in 5% sodium hydroxide for 48h. After 24 h, fragments were washed with water and transferred to lactic acid before immersing in a water bath at 95ºC until they became translucent. Layers were then photographed with an optical microscope (Motic model) coupled to a digital camera. Finally, stomatal density (SD), stomatal index (SI), stomatal polar diameter (SPD), stomatal equatorial diameter (SED), and stomatal functionality (SF) were analyzed based on procedures previously described by Batista-Silva et al. (2019) using ImageJ software (Schneider et al. 2012), in at least 10 different fields of 0.053 mm 2 per leaf. Data analysis Data were collected from experiments using a randomized block design, with each plot containing four plants per cultivar. The homoscedasticity and normality of the data were verified to meet assumptions surrounding ANOVA. If ANOVA showed significant effects, a Tukey test ( P < 0.05) was used to determine differences between cultivars. Moreover, a principal component analysis (PCA) was carried out considering both cultivars and variables. Statistical procedures were performed using R software (v. 4.3.2; R Core Team 2023). Results Water-use efficiency A significant difference between black pepper cultivars was observed in water-use efficiency (WUE). The cv. Uthirankotta (1.0 kg ha -1 mm -1 ) displayed a higher WUE average than cv. Clonada (0.2 kg ha -1 mm -1 ), reaching an efficiency of 80% higher than its counterpart (Fig. 1A). To better understand which variables contributed to such differences, we performed a principal component analysis (PCA). The outputs from PCA scaled two major components (PCA1 and PCA2), which explained about 65% of the total variation within 32 variables (Fig. 1B). Based on the PCA biplot, a remarkable divergence between cultivars was evidenced by PCA1 (52.9%). Overall, displayed clusters demonstrate a singularity for WUE and TLA, SLA and K plant in ‘Uthinrankota’, and other parameters related to water status and anatomy parameters in cv. Clonada (Fig. 1B). Gas exchange parameters Analyses of gas exchange via infrared gas analyzer revealed that cv. Clonada holds a carbon dioxide assimilation rate ( A) 30% higher than cv. Uthirankotta (Fig. 2A). Likewise, cv. Clonada showed a stomatal conductance ( g s ) 20% higher than cv. Uthirankotta (Fig. 2B). Otherwise, both transpiration ( E) and intercellular CO 2 concentration ( C i ) did not differ between cultivars (Fig. 2C, 2D). As such, no significant differences were observed for both instantaneous water-use efficiency (WUE E ) and intrinsic water-use efficiency (WUE gs ) (Fig. 2E). The photosynthetic light-response curve (A/PAR curve) was carried out under the same water availability conditions for both cultivars of P. nigrum . This assessment revealed that no significant differences in light compensation point ( l c ) and light saturation point ( l s ) were observed (Fig. 3A, 3B, 3C). On the other hand, cv. Clonada showed both A sat (22.80) and A max (29.02) higher than observed in cv. Uthirankotta (16.46 and 21.78 μmol of CO 2 m -2 s -1 , respectively) (Fig. 3D, 3E). Water status parameters Predawn water potential (Ψ pd ) did not differ when both cultivars were compared; however, cv. Uthirankotta displayed a lower midday water potential (Ψ md ) (-1.35 MPa) than cv. Clonada (-1.02 MPa) (Fig. 4A). Such responses were accompanied by a higher plant hydraulic conductance ( K plant ) in cv. Uthirankotta as compared to cv. Clonada (Fig. 4B). Parameters based on the pressure-volume (PV) curves showed that cv. Uthirankotta presents lower osmotic potential at full turgor (Ψ s100% ) (-0.93 MPa) when compared to cv. Clonada (-0.63 MPa) (Fig. 4C). Despite no significant difference was observed in osmotic potential at the turgor loss point (Ψ sTLP ), the cell bulk elastic module (Ꜫv) differed between cultivars, with cv. Clonada showing a value (17.03 MPa) 30% higher than cv. Uthirankotta (11.96 MPa). Finally, despite no difference observed in relative water content at field capacity (RWC 100% ) between cultivars, the cv. Clonada exhibited a higher relative water content at turgor loss point (RWC TLP ) compared to cv. Uthirankotta (Fig. 4D). Total leaf area (TLA) and specific leaf area (SLA) The outcomes from growth assessments demonstrated that cv. Uthirankotta presents a total leaf area (TLA) (5.76 m 2 ) higher than cv. Clonada (2.91 m 2 ), as such for specific leaf area (SLA), with an average of 121.42 cm 2 g -1 in cv. Uthirankotta and 110.98 cm 2 g -1 in cv. Clonada (Fig. S3A, S3B). Morpho-anatomical analyses Stomata were found uniformly distributed only on the abaxial side of leaves, with the occurrence of idioblasts (calcium oxalate crystals) only in cv. Uthirankotta leaves (Fig. 5A-D). The cv. Clonada exhibited a higher stomatal density (SD) stomatal index (SI), and stomatal polar diameter (SPD), compared to cv. Uthirankotta (Fig. 5E-G), while no significant difference was observed in stomatal equatorial diameter (SED) (Fig. 5G). On the other hand, cv. Clonada showed a higher leaf thickness (LT), mesophyll thickness (MT), and adaxial epidermis thickness (AdET) than cv. Uthirankotta (Fig. 6A-E), regardless no difference was observed in abaxial epidermis thickness (AbET) when both cultivars were compared (Fig. 6F). Discussion Water-use efficiency (WUE) in crop fields, estimated as the ratio of plant output (carbon assimilation rate, biomass, or yield) to water lost, is a pivotal parameter to base a sustainable production system (Briggs and Shantz 1913). In an ideal condition, the use of additional water resources can be attenuated by using plants with high WUE set up to either natural or artificial water regimes (Bramley et al. 2013; Kang et al. 2021). Therefore, understanding WUE bases and their implications for the productive responses of black pepper cultivars is fundamental for better management of water resources in irrigated fields of this crop (EMBRAPA, 2004). Here, we evidenced that cv. Uthirankotta presents a higher WUE (converting every 1 mm of water applied into 1 kg dry weight of fruit by ha), which corresponded to an efficiency of 80% higher than observed in cv. Clonada (Fig. 1A). However, no association was observed between short-term (WUE E or WUE gs ) and long-term WUE (WUE yield ) in both cultivars (Fig. 2E), and such response was closely related to differential structural and functional traits observed in growth, anatomy, physiology, and hydraulic parameters between these cultivars. Gas exchange and water-use efficiency in Piper nigrum As parameters to predict WUE, the net CO 2 assimilation rate ( A ) and transpiration ( E ) are highlighted since together they can reflect the balance of CO 2 uptake and water loss ratio (A/E) in leaves (Leakey et al. 2019; Ma et al. 2023). The last one is closely related to the diffusive water loss control mechanism which can be instantaneous (WUE E ) or intrinsic (WUE gs ) (Bacon 2004; McAusland et al. 2013). In summary, WUE yield provides a more comprehensive and long-term view based on total leaf transpiration or whole-plant (Zhang et al. 2019), while both WUE E and WUE gs reflect the immediate responses to current environmental conditions based on stomatal conductance (Flexas 2016); Herein, we have revealed that despite divergence in A (Fig. 2A), no significant differences were observed in either WUE E or WUE gs between cultivars (Fig. 2E). Collectively, it demonstrates that cv. Clonada and cv. Uthirankotta hold distinct ways to control gains of CO 2 or loss of water to meet WUE at the leaf level. Hence, whereas cv. Uthirankotta compensates a lower A with a lower stomatal conductance ( g s ), cv. Clonada buffers a higher gs with higher A (Fig. 2A). However, such responses are not reflected by the same WUE yield (Fig. 1A). Light naturally intensity fluctuations are also known to affect net CO 2 assimilation ( A ) and, consequently, WUE of most species that were moved from shade to full sun., such as Piper nigrum (Oliveira et al. 2018; Sulistyaningsih et al. 2021). In this context, light compensation point ( l c ) and light saturation point ( l s ) are two parameters related to the minimum and maximum of light to net photosynthesis (Rodríguez-López et al. 2014; Berry and Goldsmith 2019). We have evidenced that despite no differences between cultivars observed in l c and l s parameters (Fig. 3B, 3C), cv. Clonada exhibited a higher CO 2 assimilation rate at the saturation point ( A sat ) and maximum gross photosynthetic rate ( A max ) than cv. Uthirankotta (Fig. 3D, 3E). Overall, it reveals that both cultivars have distinct patterns of response to natural light variations, and thus whereas cv. Clonada can cope with higher light intensity cv. Uthirankotta reaches its maximum photosynthesis rate at a lower light intensity, which can explain at least a remarkable divergence in short-term to long-term WUE in both cultivars. Water use-efficiency and hydraulic parameters in Piper nigrum At the whole-plant level, the WUE responds directly to the way that plants buffer water status upon environmental condition variations (Bramley et al. 2013; Flexas et al. 2013). Generally, such responses are closely related to hydraulic parameters namely predawn water potential (Ψ pd ), midday water potential (Ψ md ), and plant's hydraulic conductance ( K plant ) (Yang et al. 2013). Predawn water potential (Ψ pd ) reflects soil water potential as a consequence of the balance between water uptake by roots and water loss through null transpiration - while midday water potential (Ψ pd ) in plants refers to water status at the peak of water transpiration and demand (Knipfer et al. 2020). As such, plant hydraulic conductance ( K plant ) is a measure of the ability of a plant's vascular system to transport water. It represents the flow rate of water through the plant's conducting tissues under a unit hydraulic gradient. This conductivity is crucial for understanding how efficiently plants can transport water from the soil to the leaves (Kramer and Boyer 1995; Avila et al. 2020). In our study, Ψ pd did not differ when both cultivars were compared; however, cv. Uthirankotta displayed lower Ψ md than cv. Clonada (Fig. 4A), which was accompanied by a higher K plant (Fig. 4B). Collectively, the outcomes suggest a distinct strategy between cultivars to cope with a high demand for water at midday. On the other hand, it is well known that components of the water potential such as osmotic potential at full turgor (Ψ s100% ), the osmotic potential at turgor loss point (Ψ sTLP ), bulk elastic module (Ꜫv), relative water content at field capacity (RWC 100% ) and relative water content at turgor loss point (RWC TLP ) reflect how plant regulate water status over environmental condition (Cardoso et al. 2018; Cardoso et al. 2020). We have observed that cv. Uthirankotta exhibited a lower potential at maximum turgor loss point (Ψ sTLP ) and lower bulk elastic module (Ꜫv) compared to cv. Clonada (Fig. 4C). These responses were associated with a lower relative water content at the turgor loss point (RWC TLP ) (Fig. 4D). Altogether, it is reasonable to postulate that, in contrast to cv. Clonada, the higher plant hydraulic conductance ( K plant ) of cv. Uthirankotta is related to the ability of this cultivar to keep both lower midday water potential (Ψ md ) and relative water content at the turgor loss point (RWC TLP ) without affecting gas exchange. Coupled with such responses, this cultivar also showed a lower bulk elasticity module (Ꜫv) (Fig. 4C) – which allows buffering transient reductions in the cell turgor pressure and thus keeping gas exchange and growth processes. Anato-morphological parameters and water-use efficiency in Piper nigrum Leaf morpho-anatomical modifications are crucial strategies for plants to adapt to different environmental conditions, directly influencing the efficiency of photosynthesis and the control of water loss. These changes include stomatal, mesophilic, and epidermis conductance, all essential to optimize gas exchange and improve water-use efficiency (Cal et al. 2019; Ding et al. 2020; Mitchell et al. 2013; Xiao et al. 2017; Guzmán‐Delgado et al. 2021). Stomatal index (SI), stomatal density (SD), and stomatal size (SS, stomatal polar, and equatorial diameters) are measures to characterize the structure behind gas exchange limitations. Such parameters can vary widely among plant species and are influenced by environmental factors such as light intensity, humidity, temperature, and water availability (Franks et al. 2009; Bertolino et al. 2019; Petrík et al. 2023). In general, it has been claimed that plants optimize WUE by investing in reductions in SD and mainly SS (Franks and Beerling, 2009; de Boer et al. 2012, Franks et al. 2015; Dittberner et al. 2018). Herein, cv. Clonada showed higher stomatal density (SD), stomatal index (SI), and stomatal polar diameter (SPD) compared to cv. Uthirankotta Fig. 5A-G). A prior, it reveals that cv. Clonada tends to exhibit a water loss rate higher than cv. Uthirankotta due to the increased stomatal index, size, and density per leaf area, which may affect directly WUE at the whole-plant level. As a component of leaf parameters, specific leaf area (SLA) is a trait that relates to the area of a leaf per unit leaf dry mass, which is also associated with mesophyll in terms of gas exchange and flow (Mediavilla et al. 2001; Guerfel et al 2009). Mesophyll conductance ( g m ), for instance, refers to the ease way by which CO 2 moves from intercellular spaces to the sites of carboxylation in mesophyll chloroplasts during photosynthesis and this is also influenced by the mesophyll thickness, membrane permeability, presence of intercellular air spaces and overall leaf thickness (Hassiotou et al. 2009; Flexas et al. 2012; Flexas et al. 2013). It is well established that leaves with higher SLA may have a greater proportion of spongy mesophyll tissue or present a thinner mesophyll and epidermis thickness which can contribute to increased mesophyll conductance, alleviating gas exchange resistance (Pons et al. 2009; Milla-Moreno et al. 2016; Xiong et al. 2016). In this study, cv. Clonada presented higher LT, MT, and AdET than cv. Uthirankotta (Fig. 6A-F), which was associated with lower SLA (Fig. S3). Overall, it is sensible to hypothesize that mesophyll density and thickness are overestimating CO 2 assimilation per leaf area in cv. Clonada as compared to cv. Uthirankotta and that can explain at least the lack of association between short-term (WUE E or WUE gs ) with long-term WUE (WUE yield ) in this cultivar. In close agreement with this, a higher demand for light to achieve maximum photosynthesis in cv. Clonada when compared to cv. Uthirankotta also corroborates such results concerning SLA effects on A (Fig. 3A). Conclusion This study reveals that distinct structural and functional traits observed in growth, anatomy, physiology, and hydraulic parameters underpin WUE at whole-plant levels in Piper nigrum under the optimal condition of water supply (100% field capacity). The cv. Uthirankotta shows maintenance of gas exchange at times with a higher water demand and this leads also to a higher K plant and WUE. Such responses are associated with lower adaxial stomatal density compared to cv. Clonada. On the other hand, cv. Clonada exhibits lower water-use efficiency due to a fast-stomatal closure, likely limited by the cell wall extensibility indicated by a higher bulk module elasticity. Further studies are required to investigate the divergent behavior of these cultivars under a gradient of water availability. Abbreviations A , net CO 2 assimilation rate; C a , ambient CO 2 concentration; C i , internal CO 2 concentration; E , transpiration rate; SE, standard error; SLA, specific leaf area; WUE yield , water-use efficiency at whole-plant level; WUE E , instantaneous water-use efficiency; WUE gs , intrinsic water-use efficiency; Y, yield; g s , stomatal conductance; E , transpiration; C i , intercellular CO 2 concentration; PAR, photosynthetically active radiation; I c , light compensation point; I s , light saturation point; A sat , photosynthesis rate in saturating light; A max , maximum photosynthesis rate; predawn water potential (Ψ pd ); midday water potential ( Ψ md ) plant hydraulic conductance ( K plant ); Ψ s100% , osmotic potential at full turgor; Ψ sTLP , osmotic potential at turgor loss point; Ꜫ v , bulk elastic module; RWC 100% , relative water content at field capacity; RWC TLP , water relative content at turgor loss point; TLA, total leaf area; SLA, specific leaf area; LT, leaf thickness; MT, mesophyll thickness; AdET, adaxial epidermis thickness; AbET, abaxial epidermis thickness; SD, stomatal density; SI, stomatal index; SPD, stomatal polar diameter; SED, stomatal equatorial diameter; SF, stomatal functionality; PCA, principal component analysis. Declarations Author Contributions Research conception and design: HCAS and JALJ. Investigation: HCAS, OPS, RSG, MCA, DPS, RNVR, JSM, MASG, OFL. Data analysis: HCAS and LML. Manuscript writing and proofreading: HCAS, JALJ, and LCC. Acknowledgments We gratefully acknowledge the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES) for a scholarship grantee to HCAS, the Universidade Federal Rural da Amazônia (UFRA) for core research facilities access, and Empresa de Produtos Tropicais de Castanhal LTDA (TROPOC) for financial support and field facilities. Conflict of Interests : The authors have no competing interests to disclose, either financial or non-financial. References Ahmad N, Fazal H, Abbasi BH, Farooq S, Ali M, Khan MA (2012) Biological role of Piper nigrum L. (Black pepper): A review. 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11:05:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4412806/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4412806/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61530488,"identity":"d6ede367-d60d-45fa-a209-d560a8277970","added_by":"auto","created_at":"2024-07-31 22:35:29","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":108600,"visible":true,"origin":"","legend":"\u003cp\u003eWater-use efficiency (WUE) [A] and PCA analysis [B] of two black pepper cultivars (Clonada and Uthirankotta) cultivated upon 100% field capacity. Means followed by the same letters do not differ from each other by the Tukey test (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05). Data are mean ± standard error of five replicates.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4412806/v1/baea95b1a2be7f63fc8f9cfe.jpg"},{"id":61530321,"identity":"d7d02432-2998-473d-90b3-7bf675727f3b","added_by":"auto","created_at":"2024-07-31 22:27:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":98430,"visible":true,"origin":"","legend":"\u003cp\u003eNet CO\u003csub\u003e2\u003c/sub\u003e assimilation rate (\u003cem\u003eA\u003c/em\u003e) [A], stomatal conductance (\u003cem\u003eg\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e) [B], transpiration (\u003cem\u003eE)\u003c/em\u003e [C], and intercellular CO\u003csub\u003e2\u003c/sub\u003e concentration (\u003cem\u003eC\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e) [D] in two black pepper cultivars (Clonada and Uthirankotta) cultivated upon 100% field capacity. Means followed by the same letters do not differ from each other by the Tukey test (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05). Data are mean ± standard error of five replicates.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4412806/v1/df59950b567ce5094a192588.jpg"},{"id":61530012,"identity":"f102a1e7-8ea8-4553-8215-bf99bd250c2f","added_by":"auto","created_at":"2024-07-31 22:19:29","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":98453,"visible":true,"origin":"","legend":"\u003cp\u003ePhotosynthetic light-response curve (\u003cem\u003eA\u003c/em\u003e/PAR) [A], light compensation point (\u003cem\u003el\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e) [B], light saturation point (\u003cem\u003el\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e) [C], photosynthesis rate in saturating light (\u003cem\u003eA\u003c/em\u003e\u003csub\u003esat\u003c/sub\u003e) [D] and the maximum photosynthesis rate (\u003cem\u003eA\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e) [E] in two black pepper cultivars (Clonada and Uthirankotta) cultivated upon 100% field capacity. Means followed by the same letters do not differ from each other by the Tukey test (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05). Data are mean ± standard error of five replicates.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4412806/v1/b2a407fdab40a17b3c63e7e7.jpg"},{"id":61530320,"identity":"408a66ce-3d63-4075-b098-1ab02a8f215d","added_by":"auto","created_at":"2024-07-31 22:27:29","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":128320,"visible":true,"origin":"","legend":"\u003cp\u003eOsmotic potential at maximum turgor (Ψ\u003csub\u003es100%\u003c/sub\u003e) [A], osmotic potential at turgor loss point (Ψ\u003csub\u003esTLP\u003c/sub\u003e) [A], bulk elastic module (Ꜫv) [A], relative water content at field capacity (RWC\u003csub\u003e100%\u003c/sub\u003e) [B], relative water content at turgor loss point (RWC\u003csub\u003eTLP\u003c/sub\u003e) [B], predawn water potential and midday water potential (Ψ\u003csub\u003epd\u003c/sub\u003e and Ψ\u003csub\u003emd\u003c/sub\u003e) [C] and plant hydraulic conductance (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eplant\u003c/sub\u003e) [D] in two black pepper cultivars (Clonada and Uthirankotta) cultivated upon 100% field capacity. Means followed by the same letters do not differ from each other by the Tukey test (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05). Data are mean ± standard error of five replicates.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4412806/v1/cb11662e4f0cfeed80632242.jpg"},{"id":61530011,"identity":"fb4a6933-8f72-49de-ba34-d3629132a5ee","added_by":"auto","created_at":"2024-07-31 22:19:29","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":105283,"visible":true,"origin":"","legend":"\u003cp\u003eFront view of the leaf abaxial surface [A and C, Clonada. B and D, Uthirankotta], stomatal density (SD) [E], stomatal index (SI) [F], and polar and equatorial diameter of stomata (SPD and SED) [G] of two black pepper cultivars (Clonada and Uthirankotta) cultivated upon 100% field capacity. Means followed by the same letters do not differ from each other by the Tukey test (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05). Data are mean ± standard error of five replicates. Details: 1. Stoma, 2. Guard-cell, 3*. Common epidermal cell with prismatic crystals, 3. Common epidermal cell, 4. trichome scar.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4412806/v1/2fae0703ea20e4f1f8d72df6.jpg"},{"id":61530013,"identity":"ea6f69cd-d1f9-4b16-a351-6baf509d3dc7","added_by":"auto","created_at":"2024-07-31 22:19:29","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":111883,"visible":true,"origin":"","legend":"\u003cp\u003eCross sections of leaf (AdET) [A, Clonada. B, Uthirankotta], leaf thickness (LT) [C], mesophyll thickness (MT) [D] adaxial epidermis thickness (AdET) [E], abaxial epidermis thickness (AbET) [F] of two black pepper cultivars (Clonada and Uthirankotta) cultivated upon 100% field capacity. Means followed by the same letters do not differ from each other by the Tukey test (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05). \u0026nbsp;Data are mean ± standard error of five replicates. Details: 1. Palisade parenchyma, 2. Lacunate parenchyma, 3. Xylem, 4. Phloem, 5. Fibers, 6. Secretory structure, 7. Stoma, 8. Adaxial epidermis, 9. Abaxial epidermis, 10. Trichome.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4412806/v1/d5aa81157dbafbd1d592c879.jpg"},{"id":61530662,"identity":"dbcf9343-be21-4a45-b180-7e3354077d40","added_by":"auto","created_at":"2024-07-31 22:43:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1197274,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4412806/v1/90a8a396-8ef6-4457-877c-f537e2e7791c.pdf"},{"id":61530016,"identity":"87d460ac-cca7-4e45-8280-524211ff0ca2","added_by":"auto","created_at":"2024-07-31 22:19:29","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":733136,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalData.docx","url":"https://assets-eu.researchsquare.com/files/rs-4412806/v1/80f4e306c151a96d7a47c857.docx"}],"financialInterests":"","formattedTitle":"Morpho-physiological traits associated with contrasting water-use efficiency in Piper nigrum","fulltext":[{"header":"Key message ","content":"\u003cp\u003eDistinct morpho-physiological traits rather than gas exchange at leaf levels underpin water-use efficiency in \u003cem\u003ePiper nigrum\u003c/em\u003e\u003c/p\u003e\n"},{"header":"Headings","content":"\u003cp\u003eMorpho-physiological traits related to WUE in \u003cem\u003ePiper nigrum\u003c/em\u003e\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eOriginally from tropical regions of India, the black pepper (\u003cem\u003ePiper nigrum\u003c/em\u003e) is a perennial, semi-woody, shrubby, and climbing plant, that grows attached to wooden stakes or tree trunks (Lima et al. 2010; Ahmad et al. 2012;\u0026nbsp;Rasanjali\u0026nbsp;et al. 2019). This species holds stems formed by two distinct parts: the central stem, which presents adventitious and staple-like roots (orthotropic branch), and the lateral stems, which are devoid of adherent roots and where buds sprout into flowers and fruits, thus referred to as plagiotropic branches (EMBRAPA, 2004; Serrano et al. 2012; Martins 2018). As an understory plant, \u003cem\u003ePiper nigrum\u003c/em\u003e is adapted to dappled light and temperatures between 23 and 28 \u0026deg;C, with relative air humidity ranging from 80 to 90%, thereby requiring a hot and humid climate, with total annual rainfall above 1,500 mm during the flowering and fruiting set period (Duarte et al. 2006;\u0026nbsp;Andrade, Silva,\u0026nbsp;Salles 2017). Over the recent decades, \u003cem\u003eP. nigrum\u003c/em\u003e has been cultivated in fields under full sun and this transition has impacted overall fit and yield (EMBRAPA 2004;\u0026nbsp;Oliveira et al. 2018; Sulistyaningsih et al. 2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLeaf morpho-anatomical changes comprise one of the several strategies used by plants to acclimate to different edaphoclimatic conditions (Cal et al. 2019; Ding et al. 2020). The success of such modifications is strictly associated with growth through improvements in carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) assimilation and water loss control (Mitchell et al. 2013). In this context, it is well-known that leaf morpho-anatomical changes play a crucial role in influencing photosynthesis and regulating water loss, and these modifications primarily impact resistance to gas movement (Mitchell et al. 2013; Ding et al. 2020). Among the known leaf gas flow resistances, stomatal, mesophilic, and cuticular are highlighted (Xiao et al. 2017; Guzm\u0026aacute;n-Delgado et al. 2021). Stomatal resistance refers to the resistance to gas diffusion through the stomata, while mesophyll resistance refers to the impedance to gas diffusion between the cells of the chlorophyll parenchyma, and both are directly related to anatomical characteristics of the leaf (Ding et al. 2020; Guzm\u0026aacute;n‐Delgado et al. 2021). Moreover, it has been demonstrated that leaf morphology also responds to the transition from shadow to sun in several plants (Yang et al. 2013;\u0026nbsp;Rodr\u0026iacute;guez-Lopez et al. 2014;\u0026nbsp;Sevillano et al. 2016). In this context, plants adapted to the sun typically have thicker leaves coupled to developed vascular systems compared to shade plants (Kim et al. 2005; Mathur et al. 2018). On the other hand, shade plants may have thinner leaves with less pronounced vascular structures, as they typically experience lower rates of transpiration and need to maximize light capture efficiency (Fritz et al. 2018; Mathur et al. 2018). These adaptations reflect how plants optimize their leaf morphology and physiology to efficiently use available light and water resources (Rodr\u0026iacute;guez-Lopez et al. 2014;\u0026nbsp;Sevillano et al. 2016); however, how these features impact fit and yield in \u003cem\u003eP. nigrum\u003c/em\u003e are still elusive.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWater-use efficiency (WUE) in plants is claimed as the ability of a plant to effectively use water for growth and physiological processes while minimizing water loss. It is a key determinant of plant productivity, particularly in regions with limited water availability or during periods of drought stress (Briggs and Shantz 1913;\u0026nbsp;Bramley et al. 2013; Flexas et al. 2013). WUE can be estimated in many ways, which spans either organ to whole-plant levels or short to long-term responses (Hatfield and Dold 2019;\u0026nbsp;Petr\u0026iacute;k et al. 2023). In general, divergent data have been raised in the literature when WUE at the leaf (WUE\u003csub\u003eE\u003c/sub\u003e, WUE\u003csub\u003egs\u003c/sub\u003e) and the whole-plant levels (WUE\u003csub\u003eplant\u003c/sub\u003e) are compared\u003csub\u003e\u0026nbsp;\u003c/sub\u003e(Blum 2008; Tom\u0026aacute;s et al. 2012; Medrano et al. 2015). This response is not immediately understood but has been hypothesized to be involved not only with gas exchange parameters but also with other parameters such as morphological and hydraulic traits (Yang et al.\u0026nbsp;2013;\u0026nbsp;Medrano et al. 2015;\u0026nbsp;Hatfield and Dold 2019). In this scenario, \u003cem\u003eP. nigrum\u003c/em\u003e has shown a pronounced sensitivity to drought and exhibits varied responses in WUE under such conditions (Ambrozim et al. 2022; Teles et al. 2023). However, the structural and functional aspects associated with cultivars showing distinct WUE remain to be thoroughly examined.\u003c/p\u003e\n\u003cp\u003eImproving water-use efficiency in plants is essential for sustainable agriculture, food security, and ecosystem resilience, particularly in a scenario of climate change and water scarcity (Bertolino et al. 2019; Petr\u0026iacute;k et al. 2023). Extensive efforts have been made to decipher mechanisms underlying WUE and develop strategies to improve it in both agricultural and natural systems (Gago et al. 2014; Bertolino et al. 2019; Hatfield and Dold 2019). In this sense, WUE is a determining factor for the set up and management of irrigation systems, thus reducing water loss (Yang et al 2015; Linker et al. 2016; Kang et al. 2021). However, despite the association between morpho-physiological aspects and WUE has been documented elsewhere (Dittberner et al. 2018; Bertolino et al. 2019; Ma et al. 2023; Petr\u0026iacute;k et al. 2023), there is a lack of evidence for \u003cem\u003eP. nigrum\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHerein, morpho-physiological aspects underlying water-use efficiency were investigated by assessing growth, anatomy, gas exchange, and hydraulic parameters in two cultivars of \u003cem\u003ePiper nigrum\u003c/em\u003e. Moreover, aspects related to the association between water-use efficiency at leaf and whole-plant levels are also discussed.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003ePlant material and experimental conditions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe experiment was conducted at fields of the Tropical Products Company of Castanhal (Tropoc), located in the municipality of Castanhal, PA, Brazil (01\u0026ordm; 17\u0026apos; 50\u0026rdquo; S, 47\u0026ordm; 55\u0026apos; 20\u0026rdquo; W, 40 m altitude). The study was carried out in partnership with the Study Group on Water and Soil Engineering in the Amazon (GEEASA) from the Universidade Federal Rural da Amaz\u0026ocirc;nia (UFRA). The field is composed of 2-year-old seedlings from two cultivars of \u003cem\u003ePiper nigrum\u003c/em\u003e L., namely\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ecv. Clonada and cv. Uthirankotta. They were arranged in an area of 1930 m\u003csup\u003e2\u003c/sup\u003e. The experiment is set up in a randomized blocks design, in which each plot consisted of four plants per cultivar (Clonada and Uthirankotta), in a double row, with a spacing of 4.0 m between rows and 2.20 x 2.20 m among plants. The cv. Clonada was obtained from seed germination and \u003cem\u003ein vitro\u003c/em\u003e of the Kuthiravally cultivar, an early fruit maturation material, which normally sprouts from December to February, with a period of pollination to fruit maturation of six months. It displays medium-sized foliage, purpure-colored sprouts, long ears with good filling, and medium-sized fruits, with an average production of 3 kg plant\u003csup\u003e-1\u003c/sup\u003e (Fig. S1). On the other hand, cv. Uthirankotta is a well-established cultivar in the region. It holds a late fruit maturation cycle, normally the flowering from January to March, with a phase of six months from pollination to fruit maturation. It presents wide leaves, long ears containing several rows of fruits, dark-purple-colored sprouts, and medium-sized fruits, with an average production of 3 kg plant\u003csup\u003e-1\u003c/sup\u003e (Fig. S1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe soil classification is a dystrophic Yellow Argisol (medium texture), with a predominance of secondary vegetation (Cardoso J\u0026uacute;nior et al. 2007). \u0026nbsp;The soil chemical and acidity corrections were carried out by applying liming (3.7 t ha\u003csup\u003e-1\u003c/sup\u003e) and both organic and mineral planting fertilizers as previously described by Oliveira and Nakayama (2007). Plants were cultivated under global radiation (GR) of 1303 W/m\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e(nearly 700 \u0026micro;mol de photons m\u003csup\u003e-2\u0026nbsp;\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e), and daytime temperatures of 25.3 to 26.2 \u0026plusmn; 2 \u0026deg;C (day/night), with relative air humidity between 60.5 and 86%. A total precipitation of 1,520 mm, with rainfall occurring in January (489 mm) and May (388 mm) and the lowest in February (99.8 mm)\u0026nbsp;(Fig.\u0026nbsp;S2). To confirm that all plants were kept at field capacity over the course of this experiment, the soil moisture was monitored with tensiometers installed at a depth of 20 cm-30cm. The tensiometers were positioned in line with the culture, 15 cm from the drippers. Readings were taken daily at 8:30 am. The analyses of gas exchange and water status were performed 24 months after the experiment was initiated. The yield was determined at the end of the harvest season. The entire experiment period comprised 36 months.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Yield and water-use efficiency\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFruits were harvested during their respective seasons for each cultivar (between July and October for cv. Clonada, and August to September for cv. Uthirankotta). \u0026nbsp;Based on pepper fruits fresh weight, the yield (Y) was calculated as Y=P/A, where Y, as yield (kg ha\u003csup\u003e-1\u003c/sup\u003e); P, as production (kg); and A, as area (ha). The water balance over the experiment period was monitored and expressed in mm. Thus, the water-use efficiency (WUE\u003csub\u003eyield\u003c/sub\u003e) was accomplished through the relationship between black pepper fruit productivity (kg ha\u003csup\u003e-1\u003c/sup\u003e) and water consumption (mm) (Doorenbos and Kassam\u0026nbsp;1994), as\u0026nbsp;WUE\u003csub\u003eyield\u003c/sub\u003e=Y/w, where: WUE\u003csub\u003eyield\u003c/sub\u003e, as water-use efficiency, kg ha\u003csup\u003e-1\u003c/sup\u003e mm\u003csup\u003e-1\u003c/sup\u003e; Y, as total yield, kg ha\u003csup\u003e-1\u003c/sup\u003e; w, as volume of water applied, mm.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGas exchange\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGas exchange parameters were determined using the infrared gas analyzer \u0026nbsp;(LCpro-SD, ADC BioScientific Ltd, United Kingdom). Analyzes were performed on the third to fourth fully expanded leaf of the twelfth branch from the base. The analyses included net carbon assimilation rate (\u003cem\u003eA\u003c/em\u003e), stomatal conductance (\u003cem\u003eg\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e), transpiration (\u003cem\u003eE\u003c/em\u003e), and intercellular CO\u003csub\u003e2\u003c/sub\u003e concentration (\u003cem\u003eC\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e), which were measured under photosynthetically active radiation (PAR) of 1000 \u0026micro;mol (photons) m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e s\u003csup\u003e\u0026ndash;1\u003c/sup\u003e and 400 ppm CO\u003csub\u003e2\u003c/sub\u003e at leaf level (Yin et al. 2009). Instantaneous (WUE\u003csub\u003eE\u003c/sub\u003e) and intrinsic water-use efficiency (WUE\u003csub\u003egs\u003c/sub\u003e) were estimated based on the ratio between \u003cem\u003eA\u003c/em\u003e/\u003cem\u003eE\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e/\u003cem\u003eg\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e, respectively. Response curves of net photosynthesis (\u003cem\u003eA\u003c/em\u003e) to photosynthetically active radiation (PAR) were also carried out. For this, leaves were submitted to 12 points of light intensity, varying the photosynthetically active radiation from 0 to 1400 \u0026micro;mol photons m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e via a light source (halogen bulb with 50W reflector) under a fixed concentration of 400 \u0026micro;mol mol\u003csup\u003e-1\u003c/sup\u003e air (C\u003csub\u003ea\u003c/sub\u003e) of CO\u003csub\u003e2\u003c/sub\u003e in the chamber. The photosynthetic light-response curve (\u003cem\u003eA/\u003c/em\u003ePAR curve) were adjusted as described by Lobo et al. (2013). From the \u003cem\u003eA\u003c/em\u003e/PAR curve plots, light compensation point (\u003cem\u003eI\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e), light saturation point (\u003cem\u003eI\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e), photosynthesis rate in saturating light (\u003cem\u003eA\u003c/em\u003e\u003csub\u003esat\u003c/sub\u003e), and maximum gross photosynthesis rate (\u003cem\u003eA\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e) were calculated by fitting the mechanistic model (Lobo et al.\u0026nbsp;2013).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cem\u003eWater status\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eLeaf water potential measurements were carried out at both predawn (\u0026Psi;\u003csub\u003epd\u003c/sub\u003e) and midday (\u0026Psi;\u003csub\u003emd\u003c/sub\u003e) using the Scholander pressure chamber (Scholander et al.\u0026nbsp;1965), model M 1505D (Pressure Chamber Instruments, PMS). The \u0026Psi;\u003csub\u003epd\u0026nbsp;\u003c/sub\u003ewas performed between 2:30 am and 5:30 am, while the \u0026Psi;\u003csub\u003emd\u003c/sub\u003e was carried out between 12:00 pm and 3:00 pm. Afterwards, the plant\u0026apos;s hydraulic conductance (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eplant\u003c/sub\u003e) was estimated as described by Kramer and Boyer (1995), and Avila et al. (2020), in which \u003cem\u003eK\u003c/em\u003e\u003csub\u003eplant\u003c/sub\u003e=\u003cem\u003eE\u003c/em\u003e/([-(\u0026Psi;\u003csub\u003emd\u003c/sub\u003e \u0026ndash; \u0026Psi;\u003csub\u003epd\u003c/sub\u003e)]); where\u0026nbsp;K\u003csub\u003eplant,\u0026nbsp;\u003c/sub\u003eas hydraulic conductance of the plant (mmol H\u003csub\u003e2\u003c/sub\u003eO m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e MPa\u003csup\u003e-1\u003c/sup\u003e); \u003cem\u003eE\u003c/em\u003e, plant leaf transpiration (mmol H\u003csub\u003e2\u003c/sub\u003eO m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e);\u0026nbsp;\u0026Psi;\u003csub\u003emd\u003c/sub\u003e, midday water potential (MPa);\u0026nbsp;\u0026Psi;\u003csub\u003epd\u003c/sub\u003e, predawn water potential (MPa).\u003c/p\u003e\n\u003cp\u003eParameters based on the pressure-volume (PV) curve (\u0026Psi;\u003csub\u003ew\u003c/sub\u003e x RWC) were determined on the third to fourth fully expanded leaf of the twelfth branch from the base. Two leaves were collected per plot and block, totaling 6 leaves per cultivar. Leaves were cut under water and rehydrated overnight until \u0026Psi;\u003csub\u003ew\u003c/sub\u003e reached nearly \u0026minus;0.1 MPa. \u0026nbsp;Both leaf weight and \u0026Psi;\u003csub\u003ew\u003c/sub\u003e were recorded using the Scholander pressure chamber and precision scale (de 0,001 g) over time during desiccation on the laboratory bench until \u0026Psi;\u003csub\u003ew\u003c/sub\u003e stabilization. At least four to five points were collected before and after the turgor loss point for each leaf (Tyree and Hammel 1972). Except for the first weighing (considered as FW\u003csub\u003e1\u003c/sub\u003e) of each leaf per cultivar, the others were considered as fresh weight (FW\u003csub\u003e2\u003c/sub\u003e). The leaves were dried in an oven at 65 \u0026ordm;C, and the dry mass (DW) was recorded. Then, the relative water content (RWC) at each point was calculated as RWC (%) = \u0026shy;(FW\u003csub\u003e1\u003c/sub\u003e\u0026minus; DW)∕FW\u003csub\u003e2\u003c/sub\u003e\u0026minus; DW) \u0026times; 100. Finally, the osmotic potential at full turgor (\u0026Psi;\u003csub\u003es100%\u003c/sub\u003e), osmotic potential at turgor loss point (\u0026Psi;\u003csub\u003esTLP\u003c/sub\u003e), bulk elastic module (Ꜫ\u003csub\u003ev\u003c/sub\u003e), relative water content at field capacity (RWC\u003csub\u003e100%\u003c/sub\u003e) and relative water content at turgor loss point (RWC\u003csub\u003eTLP\u003c/sub\u003e) were estimated based on recommendations of Cardoso et al. (2018) and Cardoso et al. (2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cem\u003eGrowth Parameters\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Total leaf area (TLA) was determined as described by Rhoads and Bloodworth (1964). For this, leaves were collected in the portion corresponding to \u0026frac14; of the plant height. The leaves were placed in paper bags and transferred to an oven at 65\u0026ordm; C for 72 h. Afterwards, the leaves were weighed on an analytical scale and then TLA was estimated according to Reddy et al. (1989): TLA=FW\u003csub\u003eleaf\u0026nbsp;\u003c/sub\u003ex SLA x 4.\u0026nbsp;TLA, total average leaf area of a plant (m\u003csup\u003e2\u003c/sup\u003e); FW\u003csub\u003eleaf\u003c/sub\u003e, dry weight of leaves corresponding to \u0026frac14; of the plant height (kg); SLA, specific leaf area (g m\u003csup\u003e2\u003c/sup\u003e); 4, as a constant term. The\u0026nbsp;specific leaf area (SLA) was determined as described by Hunt (1982).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cem\u003eMorpho-anatomical\u0026nbsp;\u003c/em\u003e\u003cem\u003eanalyses\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMorpho-anatomical\u003cem\u003e\u0026nbsp;\u003c/em\u003eanalyses were carried out in the median region of the fourth fully expanded leaf of the twelfth branch (from base to apex) in 3-year-old plants. Samples were immediately fixed in FAA70 (70% formaldehyde-acetic acid-ethanol) in a ratio of 1:1:18 (Johansen 1940) for 24 h and subsequently stored in 70% alcohol. This material was embedded in methacrylate (Historesin-Leica) following the manufacturer\u0026apos;s recommendations. Subsequently, samples were transversely sectioned (5 \u0026mu;m thick) with an automatic feeding rotary microtome (model RM2155, Leica Microsystems Inc., Heidelberg, Germany) and stained with toluidine blue (O\u0026apos;Brien et al. 1964).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo assess leaf thickness (LT) mesophyll thickness (MT), adaxial epidermis thickness (AdET), and abaxial epidermis thickness (AbET), the fragments of the median region of the leaves were immersed in 5% sodium hydroxide for 48h. After 24 h, fragments were washed with water and transferred to lactic acid before immersing in a water bath at 95\u0026ordm;C until they became translucent. Layers were then photographed with an optical microscope (Motic model) coupled to a digital camera. Finally, stomatal density (SD), stomatal index (SI), stomatal polar diameter (SPD), stomatal equatorial diameter (SED), and stomatal functionality (SF) were analyzed based on procedures previously described by Batista-Silva et al. (2019) using \u003cem\u003eImageJ\u0026nbsp;\u003c/em\u003esoftware (Schneider et al.\u0026nbsp;2012), in at least 10 different fields of 0.053 mm\u003csup\u003e2\u003c/sup\u003e per leaf.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cem\u003eData analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eData were collected from experiments using a randomized block design, with each plot containing four plants per cultivar. The homoscedasticity and normality of the data were verified to meet assumptions surrounding ANOVA. If ANOVA showed significant effects, a Tukey test (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05) was used to determine differences between cultivars. Moreover, a principal component analysis (PCA) was carried out considering both cultivars and variables. Statistical procedures were performed using R software (v. 4.3.2; R Core Team 2023).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003cem\u003eWater-use efficiency\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;A significant difference between black pepper cultivars was observed in water-use efficiency (WUE). The cv. Uthirankotta (1.0 kg ha\u003csup\u003e-1\u003c/sup\u003e mm\u003csup\u003e-1\u003c/sup\u003e) displayed a higher WUE average than cv. Clonada (0.2 kg ha\u003csup\u003e-1\u003c/sup\u003e mm\u003csup\u003e-1\u003c/sup\u003e), reaching an efficiency of 80% higher than its counterpart (Fig.\u0026nbsp;1A). To better understand which variables contributed to such differences, we performed a principal component analysis (PCA). The outputs from PCA scaled two major components (PCA1 and PCA2), which explained about 65% of the total variation within 32 variables (Fig.\u0026nbsp;1B). Based on the PCA biplot, a remarkable divergence between cultivars was evidenced by PCA1 (52.9%). Overall, displayed clusters demonstrate a singularity for WUE and TLA, SLA and K\u003csub\u003eplant\u003c/sub\u003e in \u0026lsquo;Uthinrankota\u0026rsquo;, and other parameters related to water status and anatomy parameters in cv. Clonada (Fig.\u0026nbsp;1B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Gas exchange parameters\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAnalyses of gas exchange via infrared gas analyzer revealed that cv. Clonada holds a carbon dioxide assimilation rate (\u003cem\u003eA)\u003c/em\u003e 30% higher than cv. Uthirankotta (Fig. 2A). Likewise, cv. Clonada showed a stomatal conductance (\u003cem\u003eg\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e) 20% higher than cv. Uthirankotta (Fig. 2B). Otherwise, both transpiration (\u003cem\u003eE)\u003c/em\u003e and\u0026nbsp;intercellular CO\u003csub\u003e2\u003c/sub\u003e concentration\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u003cem\u003eC\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e) did not differ between cultivars (Fig. 2C, 2D).\u0026nbsp;As such, no significant differences were observed for both\u0026nbsp;instantaneous water-use efficiency (WUE\u003csub\u003eE\u003c/sub\u003e) and intrinsic water-use efficiency\u0026nbsp;(WUE\u003csub\u003egs\u003c/sub\u003e)\u0026nbsp;(Fig. 2E). The photosynthetic light-response curve (A/PAR curve) was carried out under the same water availability conditions for both cultivars of \u003cem\u003eP. nigrum\u003c/em\u003e. This assessment revealed that no significant differences in\u0026nbsp;light compensation point (\u003cem\u003el\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e) and light saturation point (\u003cem\u003el\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e) were observed\u0026nbsp;(Fig. 3A, 3B, 3C). On the other hand, cv. Clonada showed both \u003cem\u003eA\u003c/em\u003e\u003csub\u003esat\u003c/sub\u003e (22.80) and \u003cem\u003eA\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e (29.02) higher than observed in cv. Uthirankotta (16.46 and 21.78\u0026nbsp;\u0026mu;mol of CO\u003csub\u003e2\u003c/sub\u003e m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e, respectively) (Fig. 3D, 3E).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eWater status parameters\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePredawn water potential (\u0026Psi;\u003csub\u003epd\u003c/sub\u003e)\u003csub\u003e\u0026nbsp;\u003c/sub\u003edid not differ when both cultivars were compared; however, cv. Uthirankotta displayed a lower midday water potential (\u0026Psi;\u003csub\u003emd\u003c/sub\u003e)\u003csub\u003e\u0026nbsp;\u003c/sub\u003e(-1.35 MPa)\u0026nbsp; than cv. Clonada (-1.02 MPa) (Fig. 4A). Such responses were accompanied\u0026nbsp;by a higher plant hydraulic conductance (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eplant\u003c/sub\u003e)\u003csub\u003e\u0026nbsp;\u003c/sub\u003ein cv. Uthirankotta as compared to cv. Clonada (Fig. 4B).\u0026nbsp;Parameters based on the pressure-volume (PV) curves showed that cv. Uthirankotta presents lower osmotic potential at full turgor (\u0026Psi;\u003csub\u003es100%\u003c/sub\u003e) (-0.93 MPa) when compared to cv. Clonada (-0.63 MPa) (Fig. 4C). Despite no significant difference was observed in osmotic potential at the turgor loss point (\u0026Psi;\u003csub\u003esTLP\u003c/sub\u003e), the cell bulk elastic module (Ꜫv) differed between cultivars, with cv. Clonada showing a value (17.03 MPa) 30% higher than cv. Uthirankotta (11.96 MPa). Finally, despite no difference observed in\u0026nbsp;relative water content at field capacity\u0026nbsp;(RWC\u003csub\u003e100%\u003c/sub\u003e)\u003csub\u003e\u0026nbsp;\u003c/sub\u003ebetween cultivars, the cv. Clonada exhibited a higher\u0026nbsp;relative water\u0026nbsp;content at turgor loss point\u0026nbsp;(RWC\u003csub\u003eTLP\u003c/sub\u003e) compared to cv. Uthirankotta\u0026nbsp;(Fig. 4D).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Total leaf area (TLA) and specific leaf area (SLA)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe outcomes from growth assessments demonstrated that cv. Uthirankotta presents a total leaf area (TLA) (5.76 m\u003csup\u003e2\u003c/sup\u003e) higher than cv. Clonada (2.91 m\u003csup\u003e2\u003c/sup\u003e), as such for specific leaf area (SLA), with an average of 121.42 cm\u003csup\u003e2\u003c/sup\u003e g\u003csup\u003e-1\u003c/sup\u003e in cv. Uthirankotta and 110.98 cm\u003csup\u003e2\u003c/sup\u003e g\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003ein cv. Clonada (Fig. S3A, S3B).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003cem\u003eMorpho-anatomical analyses\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eStomata were found uniformly distributed only on the abaxial side of leaves, with the occurrence of idioblasts (calcium oxalate crystals) only in cv. Uthirankotta leaves (Fig. 5A-D). The cv. Clonada exhibited a higher stomatal density (SD) stomatal index (SI), and stomatal polar diameter (SPD), compared to cv. Uthirankotta (Fig. 5E-G), while no significant difference was observed in stomatal equatorial diameter (SED) (Fig. 5G). On the other hand, cv. Clonada showed a higher leaf thickness (LT), mesophyll thickness (MT), and adaxial epidermis thickness (AdET) than cv. Uthirankotta (Fig. 6A-E), regardless no difference was observed in abaxial epidermis thickness (AbET) when both cultivars were compared (Fig. 6F).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWater-use efficiency (WUE) in crop fields, estimated as the ratio of plant output (carbon assimilation rate, biomass, or yield) to water lost, is a pivotal parameter to base a sustainable production system (Briggs and Shantz 1913). In an ideal condition, the use of additional water resources can be attenuated by using plants with high WUE set up to either natural or artificial water regimes (Bramley et al. 2013; Kang et al. 2021). Therefore, understanding WUE bases and their implications for the productive responses of black pepper cultivars is fundamental for better management of water resources in irrigated fields of this crop (EMBRAPA, 2004). Here, we evidenced that cv. Uthirankotta presents a higher WUE (converting every 1 mm of water applied into 1 kg dry weight of fruit by ha), which corresponded to an efficiency of 80% higher than observed in cv. Clonada (Fig. 1A). However, no association was observed between short-term (WUE\u003csub\u003eE\u003c/sub\u003e or WUE\u003csub\u003egs\u003c/sub\u003e) and long-term WUE (WUE\u003csub\u003eyield\u003c/sub\u003e) in both cultivars (Fig. 2E), and such response was closely related to differential structural and functional traits observed in growth, anatomy, physiology, and hydraulic parameters between these cultivars.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGas exchange and water-use efficiency\u0026nbsp;in Piper nigrum\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;As parameters to predict WUE, the net CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eassimilation rate (\u003cem\u003eA\u003c/em\u003e) and transpiration (\u003cem\u003eE\u003c/em\u003e) are highlighted since together they can reflect the balance of CO\u003csub\u003e2\u003c/sub\u003e uptake and water loss ratio (A/E) in leaves (Leakey et al.\u0026nbsp;2019; Ma et al.\u0026nbsp;2023). The last one is closely related to the diffusive water loss control mechanism which can be instantaneous (WUE\u003csub\u003eE\u003c/sub\u003e) or intrinsic (WUE\u003csub\u003egs\u003c/sub\u003e) (Bacon\u0026nbsp;2004; McAusland et al.\u0026nbsp;2013). In summary, WUE\u003csub\u003eyield\u003c/sub\u003e provides a more comprehensive and long-term view based on total leaf transpiration or whole-plant (Zhang et al.\u0026nbsp;2019), while both WUE\u003csub\u003eE\u0026nbsp;\u003c/sub\u003eand WUE\u003csub\u003egs\u003c/sub\u003e reflect the immediate responses to current environmental conditions based on stomatal conductance (Flexas\u0026nbsp;2016); Herein, we have revealed that despite divergence in \u003cem\u003eA\u003c/em\u003e (Fig.\u0026nbsp;2A), no significant differences were observed in either WUE\u003csub\u003eE\u003c/sub\u003e or WUE\u003csub\u003egs\u003c/sub\u003e between cultivars (Fig.\u0026nbsp;2E). Collectively, it demonstrates that cv. Clonada and cv. Uthirankotta hold distinct ways to control gains of CO\u003csub\u003e2\u003c/sub\u003e or loss of water to meet WUE at the leaf level. Hence, whereas cv. Uthirankotta compensates a lower \u003cem\u003eA\u003c/em\u003e with a lower stomatal conductance (\u003cem\u003eg\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e), cv. Clonada buffers a higher gs with higher A (Fig.\u0026nbsp;2A). However, such responses are not reflected by the same WUE\u003csub\u003eyield\u0026nbsp;\u003c/sub\u003e(Fig.\u0026nbsp;1A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLight naturally intensity fluctuations are also known to affect net CO\u003csub\u003e2\u003c/sub\u003e assimilation (\u003cem\u003eA\u003c/em\u003e) and, consequently, WUE of most species that were moved from shade to full sun., such as \u003cem\u003ePiper nigrum\u003c/em\u003e (Oliveira et al.\u0026nbsp;2018; Sulistyaningsih et al.\u0026nbsp;2021). In this context, light compensation point (\u003cem\u003el\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e) and light saturation point (\u003cem\u003el\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e) are two parameters related to the minimum and maximum of light to net photosynthesis (Rodr\u0026iacute;guez-L\u0026oacute;pez et al.\u0026nbsp;2014; Berry and Goldsmith\u0026nbsp;2019). We have evidenced that despite no differences between cultivars observed in \u003cem\u003el\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e and \u003cem\u003el\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e parameters (Fig.\u0026nbsp;3B,\u0026nbsp;3C), cv. Clonada exhibited a higher CO\u003csub\u003e2\u003c/sub\u003e assimilation rate at the saturation point (\u003cem\u003eA\u003c/em\u003e\u003csub\u003esat\u003c/sub\u003e) and maximum gross photosynthetic rate (\u003cem\u003eA\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e) than cv. Uthirankotta (Fig.\u0026nbsp;3D,\u0026nbsp;3E). Overall, it reveals that both cultivars have distinct patterns of response to natural light variations, and thus whereas cv. Clonada can cope with higher light intensity cv. Uthirankotta reaches its maximum photosynthesis rate at a lower light intensity, which can explain at least a remarkable divergence in short-term to long-term WUE in both cultivars.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eWater use-efficiency and hydraulic parameters\u0026nbsp;in Piper nigrum\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAt the whole-plant level, the WUE responds directly to the way that plants buffer water status upon environmental condition variations (Bramley et al. 2013; Flexas et al. 2013). Generally, such responses are closely related to hydraulic parameters namely predawn water potential (\u0026Psi;\u003csub\u003epd\u003c/sub\u003e), midday water potential (\u0026Psi;\u003csub\u003emd\u003c/sub\u003e), and plant\u0026apos;s hydraulic conductance (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eplant\u003c/sub\u003e) (Yang et al.\u0026nbsp;2013). Predawn water potential (\u0026Psi;\u003csub\u003epd\u003c/sub\u003e) reflects soil water potential as a consequence of the balance between water uptake by roots and water loss through null transpiration - while midday water potential (\u0026Psi;\u003csub\u003epd\u003c/sub\u003e) in plants refers to water status at the peak of water transpiration and demand (Knipfer et al. 2020). As such, plant hydraulic conductance (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eplant\u003c/sub\u003e)\u0026nbsp;is a measure of the ability of a plant\u0026apos;s vascular system to transport water. It represents the flow rate of water through the plant\u0026apos;s conducting tissues under a unit hydraulic gradient. This conductivity is crucial for understanding how efficiently plants can transport water from the soil to the leaves (Kramer and Boyer 1995; Avila et al. 2020). In our study,\u0026nbsp;\u0026Psi;\u003csub\u003epd\u0026nbsp;\u003c/sub\u003edid not differ when both cultivars were compared; however, cv. Uthirankotta displayed lower\u0026nbsp;\u0026Psi;\u003csub\u003emd\u003c/sub\u003e than cv. Clonada (Fig. 4A), which was accompanied by a higher \u003cem\u003eK\u003c/em\u003e\u003csub\u003eplant\u0026nbsp;\u003c/sub\u003e(Fig. 4B). Collectively, the outcomes suggest a distinct strategy between cultivars to cope with a high demand for water at midday.\u003c/p\u003e\n\u003cp\u003eOn the other hand, it is well known that components of the water potential such as osmotic potential at full turgor (\u0026Psi;\u003csub\u003es100%\u003c/sub\u003e), the osmotic potential at turgor loss point (\u0026Psi;\u003csub\u003esTLP\u003c/sub\u003e), bulk elastic module (Ꜫv), relative water content at field capacity (RWC\u003csub\u003e100%\u003c/sub\u003e) and relative water content at turgor loss point (RWC\u003csub\u003eTLP\u003c/sub\u003e) reflect how plant regulate water status over environmental condition (Cardoso et al. 2018; Cardoso et al. 2020). We have observed that cv. Uthirankotta exhibited a lower potential at maximum turgor loss point (\u0026Psi;\u003csub\u003esTLP\u003c/sub\u003e) and lower bulk elastic module (Ꜫv) compared to cv. Clonada (Fig. 4C). These responses were associated with a lower relative water content at the turgor loss point (RWC\u003csub\u003eTLP\u003c/sub\u003e) (Fig. 4D). Altogether, it is reasonable to postulate that, in contrast to cv. Clonada, the higher plant hydraulic conductance (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eplant\u003c/sub\u003e) of cv. Uthirankotta is related to the ability of this cultivar to keep both lower midday water potential (\u0026Psi;\u003csub\u003emd\u003c/sub\u003e)\u003csub\u003e\u0026nbsp;\u003c/sub\u003eand relative water content at the turgor loss point (RWC\u003csub\u003eTLP\u003c/sub\u003e) without affecting gas exchange. Coupled with such responses, this cultivar also showed a lower bulk elasticity module (Ꜫv) (Fig. 4C) \u0026ndash; which allows buffering transient reductions in the cell turgor pressure and thus keeping gas exchange and growth processes.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAnato-morphological parameters and water-use efficiency in Piper nigrum\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eLeaf morpho-anatomical modifications are crucial strategies for plants to adapt to different environmental conditions, directly influencing the efficiency of photosynthesis and the control of water loss. These changes include stomatal, mesophilic, and epidermis conductance, all essential to optimize gas exchange and improve water-use efficiency (Cal et al. 2019; Ding et al. 2020; Mitchell et al. 2013; Xiao et al. 2017; Guzm\u0026aacute;n‐Delgado et al. 2021). Stomatal index (SI), stomatal density (SD), and stomatal size (SS, stomatal polar, and equatorial diameters) are measures to characterize the structure behind gas exchange limitations. Such parameters can vary widely among plant species and are influenced by environmental factors such as light intensity, humidity, temperature, and water availability (Franks et al. 2009; Bertolino et al. 2019; Petr\u0026iacute;k et al. 2023). In general, it has been claimed that plants optimize WUE by investing in reductions in SD and mainly SS (Franks and Beerling, 2009; de Boer et al. 2012, Franks et al. 2015;\u0026nbsp;Dittberner et al. 2018). Herein, cv. Clonada showed higher stomatal density (SD), stomatal index (SI), and stomatal polar diameter (SPD) compared to cv. Uthirankotta Fig. 5A-G). A prior, it reveals that cv. Clonada tends to exhibit a water loss rate higher than cv. Uthirankotta due to the increased stomatal index, size, and density per leaf area, which may affect directly WUE at the whole-plant level.\u003c/p\u003e\n\u003cp\u003eAs a component of leaf parameters, specific leaf area (SLA) is a trait that relates to the area of a leaf per unit leaf dry mass, which is also associated with mesophyll in terms of gas exchange and flow (Mediavilla et al. 2001; Guerfel et al 2009). Mesophyll conductance (\u003cem\u003eg\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e), for instance, refers to the ease way by which CO\u003csub\u003e2\u003c/sub\u003e moves from intercellular spaces to the sites of carboxylation in mesophyll chloroplasts during photosynthesis and this is also influenced by the mesophyll thickness, membrane permeability, presence of intercellular air spaces and overall leaf thickness (Hassiotou et al. 2009; Flexas et al. 2012; Flexas et al. 2013). It is well established that leaves with higher SLA may have a greater proportion of spongy mesophyll tissue or present a thinner mesophyll and epidermis thickness which can contribute to increased mesophyll conductance, alleviating gas exchange resistance (Pons et al. 2009; Milla-Moreno et al. 2016; Xiong et al. 2016). In this study, cv. Clonada presented higher LT, MT, and AdET than cv. Uthirankotta (Fig. 6A-F), which was associated with lower SLA (Fig. S3). Overall, it is sensible to hypothesize that mesophyll density and thickness are overestimating CO\u003csub\u003e2\u003c/sub\u003e assimilation per leaf area in cv. Clonada as compared to cv. Uthirankotta and that can explain at least the lack of association between short-term (WUE\u003csub\u003eE\u003c/sub\u003e or WUE\u003csub\u003egs\u003c/sub\u003e) with long-term WUE (WUE\u003csub\u003eyield\u003c/sub\u003e) in this cultivar. In close agreement with this, a higher demand for light to achieve maximum photosynthesis in cv. Clonada when compared to cv. Uthirankotta also corroborates such results concerning SLA effects on \u003cem\u003eA\u003c/em\u003e (Fig. 3A).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study reveals that distinct structural and functional traits observed in growth, anatomy, physiology, and hydraulic parameters underpin WUE at whole-plant levels in \u003cem\u003ePiper nigrum\u003c/em\u003e under the optimal condition of water supply (100% field capacity).\u0026nbsp;The cv. Uthirankotta shows maintenance of gas exchange at times with a higher water demand and this leads also to a higher \u003cem\u003eK\u003c/em\u003e\u003csub\u003eplant\u003c/sub\u003e and WUE. Such responses are associated with lower adaxial stomatal density compared to cv. Clonada. On the other hand, cv. Clonada exhibits lower water-use efficiency due to a fast-stomatal closure, likely limited by the cell wall extensibility indicated by a higher bulk module elasticity. Further studies are required to investigate the divergent behavior of these cultivars under a gradient of water availability.\u003c/p\u003e\n"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eA\u003c/em\u003e, net CO\u003csub\u003e2\u003c/sub\u003e assimilation rate; \u003cem\u003eC\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e, ambient CO\u003csub\u003e2\u003c/sub\u003e concentration; \u003cem\u003eC\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e, internal CO\u003csub\u003e2\u003c/sub\u003e concentration; \u003cem\u003eE\u003c/em\u003e, transpiration rate;\u0026nbsp;SE, standard error;\u0026nbsp;SLA, specific leaf area; WUE\u003csub\u003eyield\u003c/sub\u003e, water-use efficiency at whole-plant level; WUE\u003csub\u003eE\u003c/sub\u003e, instantaneous water-use efficiency; WUE\u003csub\u003egs\u003c/sub\u003e, intrinsic water-use efficiency; Y, \u0026nbsp;yield; \u003cem\u003eg\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e, stomatal conductance; \u003cem\u003eE\u003c/em\u003e, transpiration; \u003cem\u003eC\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e, intercellular CO\u003csub\u003e2\u003c/sub\u003e concentration; PAR, photosynthetically active radiation; \u003cem\u003eI\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e, light compensation point; \u003cem\u003eI\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e, light saturation point; \u003cem\u003eA\u003c/em\u003e\u003csub\u003esat\u003c/sub\u003e, photosynthesis rate in saturating light; \u0026nbsp; \u003cem\u003eA\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e, maximum photosynthesis rate; predawn water potential (\u0026Psi;\u003csub\u003epd\u003c/sub\u003e); midday water potential (\u003cem\u003e\u0026Psi;\u003c/em\u003e\u003csub\u003emd\u003c/sub\u003e) plant hydraulic conductance (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eplant\u003c/sub\u003e); \u0026Psi;\u003csub\u003es100%\u003c/sub\u003e, osmotic potential at full turgor; \u0026Psi;\u003csub\u003esTLP\u003c/sub\u003e, osmotic potential at turgor loss point; Ꜫ\u003csub\u003ev\u003c/sub\u003e, bulk elastic module; RWC\u003csub\u003e100%\u003c/sub\u003e, relative water content at field capacity; RWC\u003csub\u003eTLP\u003c/sub\u003e, water relative content at turgor loss point; TLA, total leaf area; SLA, specific leaf area; LT, leaf thickness; MT, mesophyll thickness; AdET, adaxial epidermis thickness; AbET, \u0026nbsp;abaxial epidermis thickness; SD, stomatal density; SI, stomatal index; SPD, stomatal polar diameter; SED, stomatal equatorial diameter; SF, stomatal functionality; PCA, principal component analysis.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResearch conception and design: HCAS and JALJ. Investigation: HCAS, OPS, RSG, MCA, DPS, RNVR, JSM, MASG, OFL. Data analysis: HCAS and LML. Manuscript writing and proofreading: HCAS, JALJ, and LCC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully acknowledge the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES) for a scholarship grantee to HCAS, the Universidade Federal Rural da Amaz\u0026ocirc;nia (UFRA) for core research facilities access, and Empresa de Produtos Tropicais de Castanhal LTDA (TROPOC) for financial support and field facilities.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConflict of Interests\u003c/strong\u003e: The authors have no competing interests to disclose, either financial or non-financial.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhmad N, Fazal H, Abbasi BH, Farooq S, Ali M, Khan MA (2012) Biological role of \u003cem\u003ePiper nigrum\u003c/em\u003e L. (Black pepper): A review. 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Plant Soil 444:281\u0026ndash;298. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11104-019-04277-6\u003c/span\u003e\u003cspan address=\"10.1007/s11104-019-04277-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"acta-physiologiae-plantarum","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"acpp","sideBox":"Learn more about [Acta Physiologiae Plantarum](http://link.springer.com/journal/11738)","snPcode":"11738","submissionUrl":"https://www.editorialmanager.com/acpp/default2.aspx","title":"Acta Physiologiae Plantarum","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"black pepper, gas exchange, water status, plant hydraulic conductance","lastPublishedDoi":"10.21203/rs.3.rs-4412806/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4412806/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWater-use efficiency (WUE) also known as crop-per-drop has been the focus of several studies concerning the limitation of water and natural resources. Alongside this, morpho-physiological aspects underlying WUE in many species have been exploited to be set up to different water regimes. Here, two cultivars of \u003cem\u003ePiper nigrum\u003c/em\u003e (Clonada and Uthirankotta), growing under an irrigation system, were investigated for morpho-physiological aspects linked to WUE by accessing anatomical, morphological, photosynthetic, and hydraulic parameters. Our findings reveal that cv. Uthirankotta presents a higher water-use efficiency at the whole-plant level (WUE\u003csub\u003eyield\u003c/sub\u003e) than cv. Clonada. However, despite this difference, no association between short-term water-use efficiency (WUE\u003csub\u003eE\u003c/sub\u003e and WUE\u003csub\u003egs\u003c/sub\u003e) and long-term water-use efficiency (WUE\u003csub\u003eyield\u003c/sub\u003e) was observed for both cultivars. Such responses were instead linked to divergence in structural and functional traits observed in growth, anatomy, and hydraulic parameters between such plant materials. We believe that our report can support further studies addressing WUE in \u003cem\u003ePiper nigrum\u003c/em\u003e under contrasting water availability by assessing underlying parameters closely associated with long- rather than short-term WUE.\u003c/p\u003e","manuscriptTitle":"Morpho-physiological traits associated with contrasting water-use efficiency in Piper nigrum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-31 22:19:24","doi":"10.21203/rs.3.rs-4412806/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2024-07-07T06:30:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-14T13:18:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Acta Physiologiae Plantarum","date":"2024-05-13T07:05:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"acta-physiologiae-plantarum","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"acpp","sideBox":"Learn more about [Acta Physiologiae Plantarum](http://link.springer.com/journal/11738)","snPcode":"11738","submissionUrl":"https://www.editorialmanager.com/acpp/default2.aspx","title":"Acta Physiologiae Plantarum","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"082919a9-94b2-45c8-90ca-cdf980cd5348","owner":[],"postedDate":"July 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-08-01T10:57:04+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-31 22:19:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4412806","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4412806","identity":"rs-4412806","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}