Luana M.Luz·Ediane C.Alves·Nariane Q.Vilhena·Tamires B.Oliveira·Zara G.B.Silva·Joze M.N.Freitas·Cândido F.O.Neto·Roberto C.L.Costa·Lucas C.Costa
Abstract Plants hold biochemical and physiological mechanisms to withstand drought conditions.Generally,depending on water def icit interval,plant rehydration relies on how it can retain growth or a positive water balance-or rarely both.In this study,two species of Hymenaea,one from the Amazon and the other from the Brazilian Cerrado,were investigated for their physiological mechanism associated with growth rehydration upon short-term exposure to drought stress.Our findings demonstrate that Hymenaea courbaril tends to invest in nitrogen to the detriment of carbon compounds,-as it is limited by lower net photosynthesis -and adjust root growth to attenuate drought stress responses.In contrast,Hymenaea stigonocarpa takes advantage of higher water potential and a basal rate of lower net photosynthesis to support aboveground growth under such conditions.Hence,it is postulated that there are distinct ways of controlling water status and growth between H.courbaril and H.stigonocarpa,which are determined either by the ability of the species to keep net photosynthesis at low levels of water content or by favoring the accumulation of nitrogen compounds.Both mechanisms were effective with regards to water use efficiency and thus it is reasonable to suggest that strategies are not exclusive and may work under adverse conditions,as observed in Amazon and Brazilian Cerrado biomes.Query
Keywords Hymenaea courbaril ·Hymenaea stigonocarpa ·Growth·Photosynthesis·Water def icit
List of symbols
ΨwWater potential
ANet CO2assimilation rate
ETranspiration rate
gsStomatal conductance
WUE Water use efficiency
RWC Relative water content
RGR Relative growth rate
TDW Total dry weight
RDW Root dry weight
LDW Leaf dry weight
TCHOTotal carbohydrates
ChlChlorophyll content
As a genus of the Fabaceae family and subfamily Caesalpinioideae,Hymenaeaincludes 14 species distributed over tropical and subtropical regions (Lee and Langenheim 1975;Lorenzi 2000;Souza et al.2014;Pinto et al.2018).Among them,Hymenaea courbarilL.andHymenaea stigonocarpaMart.ex Hayne are widely dispersed in Brazil (Oliveira et al.2012;Silva and Fonseca 2016).H.courbaril,known as jatobá,is a climax,semi-deciduous and heliophyte species that may attain heights of 20-40 m,and diameters of 0.3-1.0 m (Schwartz 2018).In its natural habitat,annual rainfall ranges between 1500 and 3000 mm (De Melo and Mendes 2005).H.stigonocarpa,locally known as jatobádo-cerrado,is a late secondary tree legume (Carvalho 2007;Oliveira et al.2012),commonly occurring in the Brazilian Cerrado (Silva and Fonseca et al.2016).It will grow in dry and nutrient-poor soils but prefers well-drained land requiring an annual rainfall from 760 to 1800 mm to produce trees 20 m high and 30-50 cm diameters (Carvalho 2007).Because it produces a large amount of fruit annually,this species plays a fundamental ecological role as food for terrestrial fauna (Moraes et al.2001) and is widely cultivated to recover degraded sites.
As consequences of global climate change,shifts in rainfall regimes and time-space distribution are critical issues expected for the next decades (IPCC 2013;NASA 2014).It has been reported that periods of drought are increasing,both in the Brazilian Cerrado and Amazon regions mainly from May to September when the intensity and amount of rainfall substantially decrease (Nobre et al.2016;Alves et al.2022;Caballero et al.2022).In such conditions,forests are confronted by a critical scenario marked by wildf ires that prevent natural recovery (PRODES 2021).Based on this,reforestation in both the Cerrado and Amazon regions has been conducted with native species (Carvalho 2000).However,it is evident that such plantings must cope with a negative water balance due to high evapotranspiration coupled with low amounts of rainfall (Caballero et al.2022),which results in suboptimal conditions for plant establishment,growth,and development.
Water is the most limiting factor for both crop yields and natural ecosystem survival (Lambers et al.2008).To overcome drought stress,plants have developed mechanisms of acclimation such as osmotic and elastic adjustments (Marínde la Rosa et al.2019;Meena and Kaur 2019).Osmotic adjustment in response to soil water scarcity occurs by the active accumulation of osmolyte substances in the cells,such as polyols,amino acids,and/or sugar-like compounds (Martinez et al.2004;Anjum et al.2017).The net gain of osmolytes promotes a reduction in water potential (Ψw) in response to decreases in osmotic potential (Ψs) (Sanders and Arndt 2012;Ferchichi et al.2018;Turner 2018).Thus,the loss of turgor pressure occurs at a more negativeΨwcompared to plants that lackΨsadjustment,which allows water uptake from drier soils (Hessini et al.2009;Catuchi et al.2011;Blum 2017).Because of dehydration,the volume of plant cells can be reduced until the complete loss of turgor pressure (Martinez et al.2007;Lambers et al.2008;Moore et al.2008;Hamouda et al.2016;Rui and Dinneny 2020).The extent to which the cells can reduce their volume depends on the elasticity of their walls,which has been directly associated with tissue water storage capacitance -a variable that reflects the capacity of buffering transient alterations in water availability into the soil (Nobel 1983;Blackman and Brodribb 2011).In this regard,a higher capacitance refers to the ability to retain high turgor pressure as a function of decreases in relative water content (Koide et al.1989;Lamont and Lamont 2000 Sack and Tyree 2005).On the other hand,lower capacitance displays a wider range of water potential variation as a function of shorter shifts in cell volume (Lambers et al.2008).Nevertheless,this mechanism can prevent loss of water through stomata by achieving null pressure potential (Ψp=0) (Martinez et al.2007;Moore et al.2008;Farooq et al.2009).
It is well-known that the irreversibility of changes in plant growth and metabolism during drought stress will depend on the genotype,drought duration and severity,and stage of plant development (Jones and Corlett 1992;Furlan et al.2016).Recently,De Souza et al.(2018) unveiled the biochemical aspects involved in the rehydration capacity ofH.courbarilandH.stigonocarpaspecies under such conditions.However,adjustments in water status and metabolic shifts underlying growth and rehydration ability of these species are still to be examined.Therefore,this study investigated physiological mechanisms underlying the rehydration ability ofH.courbarilandH.stigonocarpaunder short-term exposure to drought stress by assessing growth,water relations,and metabolic parameters.
Plant material and growth conditions
The study was carried out at the Institute of Agricultural Sciences (ICA) of the Universidade Federal Rural da Amazônia (UFRA),Belém,Pará.One hundred and fifty of one-monthold seedlings of bothH.courbarilandH.stigonocarpawere purchased from the Association of the Timber Exporting Industries of the State of Pará (Aimex) in the municipality of Benevides,Pará.Seedlings were transplanted to polyethylene pots containing 18 kg of medium texture yellow latosol,previously sieved to remove impurities two months before transplanting with 1.8 g of dolomitic limestone per pot.The contents of macronutrients and micronutrients were corrected by applying 5:7.5:10 g of NPK and 0.135 g of FTE BR12 (Nutriplant®) per pot,according to overall plant demands and soil chemical and granulometric analyses (Table S1).
Plants were kept under natural light,with photosynthetically active radiation (PAR) of 1130 μmol m-2s-1,and daytime temperatures of 23 °C to 37 °C,with relative air humidity between 60.5 and 80%.Five months after acclimation,50 seedlings of each species were selected for the five experimental groups:D0,day zero -experiment beginning;D13,plants after 13 days of drought stress;D26,plants after 26 days of drought stress.Afterwards,both stressed groups were rehydrated:RH2,plants after 26 days of drought stress and 2 days of rehydration;and RH4,plants after 26 days of drought stress and 4 days of rehydration.Each group had two water regime conditions:drought stress and control (daily irrigated plants).For clearer data presentation,the control group was exclusively used for growth parameters.Every two days,pots with plants were weighed on a digital scale to check the amount of water lost by evapotranspiration,and the same volume was added to maintain the water capacity of all groups.Responses to drought stress were assessed on days zero,13,and 26,and responses to rehydration were evaluated on the 2ndand 4thdays after the period of drought stress (26 days),according to the following methods:
Plant growth analyses
After the period of drought stress imposition (D26) and rehydration (RH4),leaves,stems and roots were collected and kept in an oven at 70 °C until dry weight stabilization.Each organ was then weighed separately and total dry weight (TDW),relative growth ratio (RGR),root/shoot ratio (root:shoot) and biomass partitioning were determined as described by Hunt (1982).
Predawn water potential (Ψpd) and relative water content (RWC)
Water potential was determined in the predawn (Ψpd) period,between 4:30 and 5:30 am,using a Scholander pressure pump (PMS Instrument Co.,Corvallis,OR,USA).The analysis was performed immediately after cutting the third or fourth pair of fully expanded leaf elts.For the analysis of leaf relative water content (RWC),10 leaf discs (10 mm diameter) were removed from each plant shortly after when the frist fresh weight (FW1) was determined on an analytical scale.Discs were then transferred to Petri dishes holding 35 mL distilled water,where they remained on a bench (25 °C) for 48 h.Subsequently,discs were placed on filter paper to remove excess water and were weighed to determine the turgid weight (FW2) .Discs were then transferred to a paper bag and placed in an oven between 65 and 70 °C for 48 h.Finally,the dry weight of the discs (DW) was determined.The leaf relative water content was calculated as described by Slavick (1979) as:
Leaf capacitance
Leaf capacitance (C) was estimated from the reciprocal slope of the ΔΨpd/ΔRWC relationship according to Nobel (1983) and Blackman and Brodribb (2011).This parameter was also expressed as % water loss MPa-1.
Gas exchange
The rates of net photosynthesis (A),stomatal conductance (gs),transpiration (E),and water use efficiency (WUE) -as a ratio ofA/E-were determined using an infrared gas analyzer (model LCi 6400,Hoddesdon,UK).Measurements were taken between 09:00 and 10:00 am.Gas exchanges were measured when the density of photosynthetic photon flow was higher than 1000 μmol m-2s-1.The average vapor pressure def icit between the air and the leaf ranged from 2.6 to 3.7 kPa,while the average leaf temperature was from 34 °C to 39 °C.Measurements were made on expanded leaves of the third or fourth pair from the shoot tip.
Determination of nitrate reductase (NR) activity
NR activity was determined as described by Hageman and Hucklesby (1971).Approximately 200 mg of leaf discs with 0.5 cm diameter were placed in test tubes containing 5 mL of phosphate-buffered saline (0.1 M,pH 7.4,Sigma Aldrich®),1% isopropanol (v/v),and KNO3,and subsequently covered with aluminum foil (dark treatment).The tubes were submitted to vacuum for 2 min and then placed in a bath at 30 °C for 30 min under dark conditions.In test tubes,1 mL aliquots of phosphate-buffered saline (0.1 M,pH 7.4,Sigma Aldrich®)+2 mL of the diluted extract+1.0 mL of 1% sulfanilamide+1.0 mL of N-1-naphthylethylenediamine dihydrochloride (NNEDA) 0.02%,were added for a total final volume of 5 mL,and then incubated for 15 min.The readings were made on the spectrophotometer at 540 nm.The result of NR activity was estimated through NO2-production in the reaction assay,expressed in mmoles of NO2-g FW-1h-1,from a standard curve obtained with KNO2(ACS reagent,≥ 96.0%,Sigma-Aldrich®).
Determination of total soluble carbohydrates content (TCHO)
Approximately 50 mg dry weight of leaves were placed in test tubes with 5 mL of distilled water and kept in a water bath for 30 min at 100 °C.The samples were then centrifuged in a bench-top centrifuge (1000 rpm) for 10 min,and 0.5 mL of 5% phenol and subsequently 2.5 mL of concentrated H2S O4added.Finally,the test tubes were placed on a bench for 20 min before readings at 490 nm in a spectrophotometer.For the calculation of total soluble carbohydrates,a standard glucose curve was used,and the results expressed in mmol of glucose g DW-1as described by Dubois et al.(1956).
Determination of total free amino acids content (AA)
Approximately 50 mg of leaf tissue were weighed and placed in test tubes with 5 mL of water distilled,hermetically sealed and incubated in a water bath at 100 °C for 30 min.The test tubes were then centrifuged at 6,000 rpm for 10 min.After extraction,the supernatant was collected.Extract amounts of 0.1 mL were placed in test tubes containing 0.4 mL distilled water,and 0.25 mL of acetate buffer solution (0.1 M,pH 5.0,Sigma Aldrich®) added.After shaking,tubes were hermetically sealed and placed in a water bath for 15 min at 100 °C.Subsequently,the reaction was stopped in an ice bath,when 1.5 mL of 50% (v/v) ethanol solution was added.After the test tubes were kept for 20 min at room temperature,readings were performed in a spectrophotometer at 570 nm.The contents of total free amino acids were determined based on a standard curve adjusted from increasing concentrations of standardized L-glutamine amino acids,and the results expressed in μmol of AA g DW-1as described by Peoples et al.(1989).
Determination of chlorophyll content (Chl)
Four leaf discs were removed with a manual punch 11 mm in diameter,weighed and placed in screw-top tubes containing 5 mL of N,N-dimethylformamide at 4 °C in the dark.After ten days,the chlorophyll content was determined by the N,N-dimethylformamide method (Inskeep and Bloom 1985) in a spectrophotometer,with readings at 647 nm and 664.5 nm.
Data analysis
The data were obtained from a completely randomized design in which was composed of two species,five treatments,five replications and two water regime conditions,totaling 100 experimental units.The data were previously evaluated for normality by Shapiro-Wilk test (P＞ 0.05),and percentages of biomass partitioning were square rootarcsine transformed to meet general assumptions.Significant differences between species,or between treatment and respective control,or time of evaluation,were tested using Student’s t-tests assuming equal variance.For metabolic compounds throughout the time of evaluation,the described differences were systematically statistically grounded,based on ANOVA,whereP＜ 0.05 was considered significant.If ANOVA showed significant effects,the Tukey’s test was used to determine differences among days.Regression analysis was also performed to integrate key gas exchange variables for each species.Specifically forAandgs,a correlation analysis was carried out.All analyzes were accomplished with R software version 4.1.0 (R Foundation for Statistical Computing,Vienna,Austria).
The analyses of growth parameters revealed that drought stress decreased TDW and RGR in both species ofHymenaeaeven after rehydration period (RH4) (Fig.1 A,B).Values ranged from 42 to 23 g,respectively,forH.courbaril,and from 11 to 6 g,respectively,forH.stigonocarpa,thereby showing a decrease of 44% in TDW and RGR in both species compared to their controls.However,H.courbarilincreased root:shoot ratios by 7% in contrast to decreases of 4% byH.stigonocarpa(Fig.1 C).Such results were closely related to shifts in biomass partitioning,in whichH.courbarilshowed a significant 14% increase in RDW (root dry weight).H.stigonocarpareduced RDW by 6% and simultaneously increased LDW (leaf dry weight) by 17% (Fig.1 D).
Fig.1 Growth parameters of H.courbaril and H.stigonocarpa under 26-day drought stress and their respective controls; A:total dry weight,B:relative growth rate (RGR),C:root:shoot ratio,D:biomass partitioning.Asterisks indicate values determined by the Stu-dent’s t-test to be significantly different from the controls (P ＜ 0.05) within the same species.Data are mean ± standard error of five replicates
Given that plant biomass is a result of photosynthesis rate,the photosynthetic performance ofHymenaeaspecies under drought stress conditions were examined using a portable infrared gas analyzer.Such assessments demonstrate that periods of drought stress and rehydration significantly affect gas exchange regardless of species,but the overall responses were notable forH.courbaril(Fig.2).Decreases inAwere observed over the drought stress period,reaching the lowest values on the 26thday (D26) (Fig.2 A).InH.courbaril,drought stress conditions decreasedAup to six times more than inH.stigonocarpaover the same period of evaluation (Fig.2 A).
Notably,responses observed inAwere accompanied by decreases ings(Fig.2 A and B).Values ranged between 201 and 26 mmol m-2s-1on the 26th day of drought stress (D26) inH.courbaril(Fig.2 B),reflecting decreases 3 -4 times greater than observed inH.stigonocarpa.In agreement with stomatal behaviour,Ewas also affected.Both species decreasedEfrom the 13thday of stress (D13),reaching low values on D26 (Fig.2 C).H.courbarilandH.stigonocarpareduced transpiration by 60% and 93%,respectively,after day 26 (D26) of drought stress.However,despite alterations in net photosynthesis,there were no significant differences in WUE throughout the evaluation period for both species (Fig.2 D).
Fig.2 Gas exchange parameters in H.courbaril and H.stigonocarpa under drought stress throughout the time of treatment imposition (D0 -D26,days of dehydration;RH2 -RH4,days of rehydration).A:photosynthesis (A),B:stomatal conductance (gs),C:transpira-tion (E),D:water use efficiency (WUE).Asterisks indicate values determined by the Student’s t-test to be significantly different from D0 (P ＜ 0.05) within the same species and time of analysis.Data are mean ± standard error of five replicates
To assess plant water status,Ψpd(pre-dawn water potential) and RWC (relative water content) were measured.Ψpddecreased significantly as a function of water stress interval in bothHymenaeaspecies,with more notable responses inH.courbaril(-2.5 MPa) compared toH.stigonocarpa(-0.94 MPa) (Fig.3 A).BothH.courbarilandH.stigonocarparecovered their initial water potentials,with averages of -0.45 and -0.35 MPa,respectively (Fig.3 A).However,whileH.stigonocarpafully recovered after 2 days of rehydration (RH2),H.courbarilreached its initial water potential only after 4 days (RH4) (Fig.3 A).RWC also showed a wide variation for the two species over the drought stress period,ranging from 70 to 92% (Fig.3 B).Similarly,H.stigonocarpasignificantly recovered its initial RWC (92%) after 2 days of rehydration (RH2) (Fig.3 B);H.courbarilreached its initial water content only after 4 days of rehydration (RH4) (Fig.3 B).
Fig.3 Plant water status of H.courbaril and H.stigonocarpa under drought stress throughout the time of treatment imposition (D0 -D26,days of dehydration;RH2 -RH4,days of rehydration).A:water potential (Ψpd),B:relative water content (RWC).Asterisks indi-cate values determined by the Student’s t-test to be significantly different from D0 (P ＜ 0.05) within the same species and time analysis.Data are mean ± standard error of five replicates
Regression analysis showed that gas exchange (A,gs) decreased as a function of a negative water potential gradient (Fig.4 A,B).However,these parameters were less affected inH.stigonocarpacompared toH.courbaril,which showed a decrease of 25% and 20% inAandgs,respectively (Fig.4 A,B).Interestingly,when the relationship betweenΨpdand RWC was examined,there was an abrupt loss of linearity in the regression model forH.courbaril(Fig.4 C).This was accompanied by a significant difference in leaf capacitance between species (Fig.4 C).InH.courbaril,the rate of water potential change as a function of RWC was significantly higher (slope=-0.040) compared to that forH.stigonocarpa(slope= -0.026) (Fig.4 C).This suggests thatH.stigonocarpamay lose up to 35% more water thanH.courbarilwithout altering water potential (Fig.4 C).Finally,as expected,there was a positive correlation betweenAandgsand the coeffi-cients of correlation were 0.95 and 0.94,respectively,forH.courbarilandH.stigonocarpa(Fig.4 D).
To investigate shifts in metabolic events,levels of TCHO,AA,NR (nitrate reductase) activity,and Chl(chlorophyll content) were also evaluated over drought stress and rehydration periods (Table 1).There was an overall increased TCHOinH.stigonocarpabecause of drought stress but not inH.courbaril(Table 1).Increases inH.stigonocarpareached 2.8 mmol glucose g-1DW,three times the levels observed in plants under 100% of water availability.Despite the high variation,H.stigonocarpasignificantly recovered its initial TCHOlevels within four days of rehydration (RH4) (Table 1).On the other hand,a continuous drought stress significantly increased AA level inH.courbarilbut not inH.stigonocarpa(Table 1).Variations of AA inH.courbariloccurred from 49.2 to 84.2 μmol amino acid g-1DW until the final day of stress (D26) (Table 1).Moreover,this species also showed a significant increase in AA (41%) and a decrease in Chl(-40%) over the drought period,a different pattern fromH.stigonocarpaspecies where there were no alterations detected (Table 1).As a link for carbon and nitrogen metabolism,NR activity displayed an opposite behaviour regarding amino acid content.Decreases in NR activity were observed over 26 days of drought stress (D26) and 2 days of rehydration (RH2) inH.courbaril,with no significant differences inH.stigonocarpa.Over this period,H.courbarilreduced NR activity to less than half of its initial activity to 28% (Table 1).
Plants possess biochemical and morphophysiological mechanisms to maintain growth and water balance during drought conditions (Chakhchar et al.2017,2018;Gupta et al.2020).Among them,increased water uptake from the soil,reduced water loss by stomatal control,tissue water capacitance adjustment,and antioxidant defense system activation are notable (Chakhchar et al.2015,2016;Gupta et al.2020).In this study,there was evidence that species ofHymenaea,one from the Amazon and other from the Brazilian Cerrado,have distinct physiological mechanisms to overcome shortterm drought stress (26 days).Overall,such mechanisms were closely related to full rehydration after 2 or 4 days and were determined by either the ability of the species to keep the basal rate of net photosynthesis at low water content or by accumulating nitrogen-like compounds.Interestingly,regardless of the mechanism,bothH.courbarilandH.stigonocarpashowed an effective strategy concerning water use efficiency.
Plant growth has been noted as the first process to be affected by drought,with a consistent penalty to TDW (Liu and Stützel 2004;Hessini et al.2008,2009;Erice et al.2010;Feng et al.2016;Al-Yasi et al.2020).In such conditions,the positive pressure required to drive cell expansion and plant growth may be zero (Peaucelle et al.2012;Cosgrove 2016a,2016b).Moreover,it is known that changes in water pressure occur primarily in the aboveground portions of the plant in deference to root tissues because the water potential gradient reduces along the root-to-shoot plant body (Erice et al.2010;Brunner et al.2015;Robbins and Dinneny 2015).A distinct pattern of fitting and growth was observed betweenHymenaeaspecies;H.courbarilshowed the same TDW loss (Fig.1 A) as compared toH.stigonocarpa;however,it had a higher root dry weight (Fig.1 D).H.stigonocarpa,on the other hand,maintained net photosynthesis at base levels which supported aboveground growth maintenance (Fig.1 D).These findings suggest an occurrence of different growth patterns betweenH.courbarilandH.stigonocarpaunder drought stress over short intervals.
Stomatal behaviour is closely influenced by plant water status and cell wall composition (Shtein et al.2017;Al-Yasi et al.2020).Kim and Lee-Stadelman (1984) studied osmotic adjustment,cell wall bulk elastic modulus and stomatal behaviour during and after drought stress and rehydration inPhaseolus vulgarisL.They demonstrated that the sensitivity of the primary leaf to drought stress is related to lower cell wall elasticity,which demonstrated a high relative water content at the turgor loss point and thus a lower tissue capacitance.Similarly,Blackman and Brodribb (2011) found a negative correlation between leaf capacitance and cell wall lignin content which impedes cell walls to maintain positive pressure.In the present study,reductions inAandgsoccurred in response to variations in pre-dawn water potential (Ψpd) in both species (Fig.4).However,in the same range ofΨpdvariation,H.courbarilshowed a marked decrease in stomatal conductance (gs) compared toH.stigonocarpa.In conf irmation of this,a marked decrease inΨpdwas observed inH.courbarilin the same RWC variation range,which was not observedin H.stigonocarpa.In this sense,considering that a high tissue capacitance maintains positive turgor pressures at low RWC (Blackman and Brodribb 2011;Le Gall et al.2015),it is reasonable to assume thatH.stigonocarpahas a higher leaf capacitance compared toH.courbaril,which underpins growth and gas exchange maintenance in such conditions.
Fig.4 Relationship between plant water status and gas exchange parameters in H.courbaril and H.stigonocarpa under drought stress.Regression or correlation coefficient is shown when P ≤ 0.001 (***),P ≤ 0.01 (**),P ≤ 0.05 (*) and P ≤ 0.1 (+).Data are mean ± standard error of five replicates
Metabolism is coupled with the capacity of plants to resist overall stresses (Anjum et al.2017;Ferchichi et al.2018;Turner 2018).The response ofH.courbarilto carbon starvation in drought conditions has been reported by Brito et al.(2016) and De Souza et al.(2018).Evidence of mechanisms underlying the metabolic response to drought inHymenaeaspecies was provided by De Souza et al.(2018).They observed an accumulation of osmotic compounds over the drought and rehydration periods for bothH.courbarilandH.stigonocarpaspecies.This osmotic control explains,at least partially,the capacity of both species to resist adverse conditions of drought.In this study,it has been further shown thatH.courbarilcompensates for the loss of photosynthesis products by accumulating nitrogen compounds (Table 1) and modulating root growth (Fig.1 C and D).At the same time,H.stigonocarpamaintains a base level of net carbon assimilation (Fig.2 A) and aerial growth (Fig.1 D) to reduce drought stress.Together,it illustrates a distinct way of controlling water status and growth byH.stigonocarpaandH.courbaril,determined either by the capacity of the species to keep net photosynthesis at lower water content or by favouring accumulation of nitrogen compounds.In agreement with such behaviour,it is well-known that nitrate reductase activity is controlled at the level ofgene expression (MacKintosh and Meek 2001;Yanagisawa 2014),being markedly downregulated by NH4+,glutamine and other amino acids (Oaks et al.1977;Srivastava 1980;McCarty and Bremer 1992).In our study,the lowest NR activity inH.courbarilwas accompanied by increases in total free amino acid levels after drought stress (RH2),which suggests a likely occurrence of a negative response to AA accumulation,not observed inH.stigonocarpa.
AcknowledgementsWe gratefully acknowledge the Universidade Federal Rural da Amazônia (UFRA) and Museu Paraense Emílio Goeldi forf inancial support and core research facilities access.
Author contributionsResearch conception and design:LML and RCLC.Investigation:LML,ECA,NQV,TBO,and JMNF.Data analysis:LML,RCLC,CFON,and LCC.Manuscript writing and proofreading:LML,ZGBS,and LCC.
This study has provided evidence of distinct physiological mechanisms underlying growth and rehydration ability following short-term exposure to drought in two contrasting species ofHymenaea,one from the Amazon and the other from the Brazilian Cerrado (Fig.S1).Overall,it was shown thatH.courbarilinvests in nitrogen to the detriment of carbon compounds,as it is limited by lower photosynthesis,and modulates root growth to reduce drought stress.H.stigonocarpa,however,takes advantage of higher water potential and a base rate of net photosynthesis to support aboveground growth in such conditions.Consequently,it is reasonable to postulate that there are diverse ways of controlling water status and growth byH.stigonocarpaandH.courbarilwhich are determined either by the ability of the species to maintain photosynthesis at lower water contents or by favoring nitrogen compound accumulation.Both mechanisms provide an effective strategy for water use efficiency.Therefore,it is sensible to suggest that such strategies are not exclusive and may work under different edaphoclimatic conditions,as observed in the Brazilian Cerrado and Amazon biomes.
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