Resource generation is critical to sustain moisture stress in Melia dubia Cav.

doi:10.21203/rs.3.rs-5063430/v1

The present study is an effort to understand the role of photosynthesis in enduring moisture stress in Melia dubia. Five germplasm lines with the highest and lowest growth from a pool of forty-two lines were selected based on their growth performance under rainfed conditions. The carbon assimilation rate of germplasm lines with the highest and lowest growth ranged between 18.7 and 15.8 µmol m^− 2s^− 1 and 15.63 and 12.30 µmol m^− 2s^− 1 at field capacity, respectively. However, it reduced (14.47 to 9.87 µmol m^− 2s^− 1 and 9.53 to 6.07 µmol m^− 2s^− 1, respectively) when subjected to 50% moisture stress. The mean reduction was 29.41% and 44.30%, respectively. Similarly, the germplasm lines with higher growth showed a lesser reduction in biomass when exposed to moisture stress, both in nursery and field experiments. The higher stomatal conductance in germplasm lines with higher growth, even under moisture stress, can be due to an anisohydric stomatal response. Both diffusive and carboxylation processes of photosynthesis were higher in the germplasm lines with the highest growth. The photosynthetic rates and growth of better-performing lines under moisture stress suggest that maintaining carbon assimilation during such conditions enhances stress tolerance. Furthermore, germplasm lines that perform better under moisture stress showed efficient partitioning of assimilated carbon among the shoot and root systems. These findings imply that increased carbon assimilation during moisture stress contributes to improved fitness for enduring such conditions, thereby indicating photosynthesis as a trait for early prediction of moisture stress tolerance at the seedling stage.

Photosynthetic rates

Moisture stress

Drought tolerance

Melia dubia

Moisture stress is the most important deterrent to plant productivity. Approximately one-third of the world’s arable land is expected to suffer chronically from inadequate water by 2050 (Wallace 2000). India, with 67% of its arable land under rainfed agriculture (Venkateshwarulu 2019) and 75% of it under arid and semiarid conditions (Singh and Joshi 1979), is considered to be one of the most vulnerable countries for climate change (IPCC 2007; Seager et al. 2007). In India, 32% of the land is degraded, and 25% of the degraded land is undergoing desertification (Gore 2022). These changes would put both food security and environmental safety at risk in India and other arid and semiarid regions of the world. Thus, vulnerability to rainfall due to climate change leading to drought is a major concern, especially in arid and semiarid regions (Peel et al. 2007).

Post-Kyoto Protocol, climate change discussions have been more oriented towards improving carbon sequestration in terrestrial vegetation systems (IPCC 2007). With the continuous increase of GHG in the atmosphere (NOAA 2022) and changes in land use systems across the globe (Doos 2002), there is an urgent need to increase green cover through reviving low-productive marginal lands, croplands, and forests to mitigate climate change (Yang et al. 2020). With various measures available to improve the global environment, carbon sequestration using trees is an ecologically and economically viable option as it provides sustained yield of energy, fiber, and lumber along with the largest sustained climate mitigation process in a cost-effective manner (Stern 2007). Trees having distinct characteristics of secondary growth and a long life span stand out from annuals in drought tolerance, growth, and carbon stocking (Marie et al. 2013; Manzoni et al. 2014). However, to establish tree cover effectively, especially under rainfed conditions, it is imperative to identify drought-tolerant tree species.

The plant's response to drought is a complex phenomenon. Plant species acclimatize and adapt to stressful environments predominantly by optimizing growth (Davies et al. 2011). Though a rich literature exists on plant responses to moisture stress, solutions to address it still remain elusive (Bréda et al. 2006; MacDowell, 2008). Plants respond to moisture stress through a variety of morphological manifestations (Devakumar et al. 1999) to manipulate functional responses (Maire et al. 2013) under moisture stress. The response varies with the intensity of stress and the ensuing dynamics of plant-environment relations (Manzoni et al. 2014). Variations seen in growth under stress reflect inherited abilities (Zobel and Tolbert 1984), whose expressions are driven by the surrounding environment (Bre´da et al. 2006). Various adaptive mechanisms to sustain growth and survive are reported among and within plant species (Sade et al. 2012).

Photosynthesis and transpiration are the most critical physiological responses under moisture stress because these two functions are often mutually exclusive. Therefore, performance under moisture stress depends on the way these two processes coordinate (Lawson and Blatt 2014), which is expressed as water use efficiency (WUE). The mechanisms through which these two functions coordinate is believed to be mainly through isohydric and anisohydric stomatal responses (Attia et al. 2015; MacDowell 2008).

The genotypic variations in WUE are found to exist among and within the species (Attia et al. 2015), which are regulated by atmospheric and intercellular water and CO₂ vapour pressure differences (Hubick and Farquhar 1989). The regulation of WUE is possible either by reducing transpiration rates when there is a drop in soil water potential or by an increase in atmospheric demand. This also reduces photosynthesis and is seen in plants with anisohydric stomatal behaviour. The second approach is to maintain steady stomatal conductance irrespective of soil water potential and atmospheric demand to maintain photosynthesis, which is referred to as isohydric stomatal response (Tardieu and Simonneau 2013). Among these two approaches, plants with isohydric behaviour are reported to be more drought-tolerant than isohydric types (McDowell et al. 2008). Isohydric behavior, although not a water conservation approach, helps in generating more resources and reduces the risk of carbon starvation-related processes (McDowell et al. 2008), thereby improving the fitness of plants to deal with moisture stress.

Thus, even small changes in photosynthetic rates can substantially increase plant growth and biomass over the course of a growing season (Zhu et al. 2007), especially under stress. Similarly, small changes in transpiration rates can make a noticeable difference in carbon and water cycles (Betts et al. 2004). Therefore, the aim of the present study is to understand the genotypic differences in carbon assimilation that influence growth performance in Melia dubia. This species is one of the fast-growing, drought-tolerant tree species (Tolia et al. 2018; Warrier 2011; Parthibhan et al. 2009) popular in semiarid subtropical regions of southern India. It is seen as a monoculture plantation as well as an agroforestry tree component. Identifying germplasm lines with better drought tolerance for this tree species can help sustain livelihoods and restore the ecosystem in the semiarid region. Further, understanding the carbon assimilation trait can help reduce the complex physiological and morphological responses into simple plant traits that can be used in tree improvement programs.

The present study was conducted in the University of Agricultural Sciences, GKVK, Bangalore, India, located at 12°58ʹ N, 77°35ʹ E, at an altitude of 930 meters above mean sea level. Ten germplasm lines of Melia dubia were selected after a field evaluation of forty-two lines for four years (half of the rotation period) at Hoskote by Karnataka State Forest Department, located around 30 km southwest of Bangalore (13°52ʹ N latitude and 77°52ʹE longitude, 875 above MSL). According to the Central Ground Water Board report (2013), the normal average annual rainfall of Hoskote is 775 mm (1901–1970), but in recent years, it has been much less (Online resource 1). It is one of the 135 (out of 186) taluks in Karnataka which was declared drought-affected for three successive years from 2012.

Five out of ten germplasm lines selected for this study recorded the highest growth in the field (MD058, MD013, MD111, 268 and 262) and five recorded the lowest growth (MD126, 69, 270, MD115 and MD117). These ten lines were selected for the present study (Online resource 2). The selected lines were vegetatively propagated using the softwood cutting method (Warrier 2012) and used in this study.

Lysimeters of 40 kg capacity were filled with potting mixture (red earth + organic matter + sand in 3:1:1 proportion) and were used in the experiment. Ten plants from ten germplasm lines were maintained at field capacity (FC), and a similar set of plants were maintained at 50% of field capacity (50% FC is considered as 50% moisture stress). Uniform seedlings were planted one each in the lysimeter and allowed to establish for 30 days.

In order to maintain the above-mentioned two moisture regimes, empty lysimeters were weighed (W1) and filled with a 40 kg potting mixture and then weighed (W2). The lysimeters with the potting mixture were saturated with water, and excess water was allowed to drain off from the taps provided at the bottom. After 48 hours, again, the lysimeters were saturated with water to ensure that the soil is completely saturated with water and weighed after another 24 hours (W3). The weight of the lysimeters with the potting mixture after the next 24-hour cycle is considered as the weight of lysimeters at field capacity. The difference in weight between W3 and W2 of each lysimeter is considered as the amount of water required to maintain the moisture levels at field capacity (Udayakumar et al. 1998). Irrigation was gradually reduced in one set of lysimeters till the moisture levels were reduced to 50% of the field capacity and maintained till the end of the experiment (considered as 50% moisture stress treatment). After attaining 50% of the FC, both sets of plants were exposed to two moisture regimes (FC and 50% FC) for 41 days and harvested. Individual lysimeters were weighed at 24-hour intervals and moisture was replenished to maintain the set moisture levels.

Gravimetric approach for assessing the water use efficiency (WUE)

The exposed soil surfaces of the lysimeters were covered with pebbles to minimize the evaporative losses from the exposed soil surface. They were kept under a rainout shelter and weighed every day between 10 and 11 A.M. to measure the moisture loss over 24-hour cycles and the lost moisture was replenished to maintain the desired soil moisture status in each lysimeter. The amount of water added to each lysimeter is considered as the water transpired. Total amount of water added to each lysimeter over 41 days is considered as the cumulative water transpired (CWT) and mean transpiration rate (MTR) is derived by computing the ratio of CWT to the functional leaf area (LAD). The MTR is a time integrated measure of water loss through transpiration process. The WUE is derived using the ratios of the total dry matter (TDM) produced during the experimental period to the CWT.

Growth assessment

The initial plant height, leaf area, and biomass were recorded at the time of transplanting as well as at the end of the experiment. The leaf area was measured using an Image analysis system (WinDiad-3, Delta-T devices Ltd, UK). Leaf, stem and roots were segregated and oven-dried at 70°C till a constant weight was obtained between the two consecutive weightings. The root-to-shoot ratio, leaf area duration (LAD) and net assimilation rate (NAR) were derived from these primary growth parameters. The LAD was computed (Atwell et al. 1999) to assess the active photosynthetic surface area that prevailed during the experimental period. The NAR is the net cumulative carbon gained from the photosynthetically active leaf surface area. The drought susceptibility index (DSI) was derived from the modified formula given by Fischer and Maurer (1978) and Winter et al. (1988). In the said modification, the total biomass produced per plant under two moisture regimes of field capacity and 50% moisture stress is used in place of grain yield. Root volume was measured based on the volume displacement method. A graduated measuring cylinder was filled with a known volume of water, and the root portion of the harvested plant was immersed into the cylinder. A change in the volume displaced is considered as the volume of the root. Specific leaf area (SLA) was derived using the leaf area of a fresh leaf, divided by its oven-dry mass.

Photosynthesis measurement

The carbon assimilation rate is assessed using three different models such as the instantaneous CO₂ exchange method (A), the cumulative carbon assimilation response using the stable carbon isotope discrimination approach (∆¹³C) and the third one is the conventional approach based on biomass accumulation (NAR).

The instantaneous CO₂ gas exchange measurement was made using a portable photosynthesis system (CIRAS-3, PP systems, USA). Thirty days after the initiation of moisture stress measurements were made on well-exposed and fully matured leaves, between 9 A.M. and 11 A.M. Photosynthetic parameters were measured at photosynthetic photon flux density (PPFD) of 1500 µmol m^− 2 s^− 1 and block temperature was maintained at 28°C. Light and CO₂ response curves were also developed using the same device by altering the light intensities (artificial using a light source of the device) and CO₂ concentrations (using a pure CO₂ cartridge). The light intensity and the CO₂ concentrations were increased from lower to saturation levels. The assimilation rate was plotted against the light intensities and CO₂ concentrations with best-fit polynomial function to develop light and CO₂ response curves using portable photosynthesis system (CIRAS-3, PP systems, USA).

Stable carbon isotope in the plant tissue (Δ¹³C) is considered to be one of the reliable surrogates for cumulative response of photosynthesis and stomatal conductance (Francy and Farquhar 1982; Brienen et al. 2011). Δ¹³C isotope estimation was made using leaf samples drawn at the end of the experiment from plants grown at two moisture regimes. Approximately 1 mg of finely ground leaf samples were packed in silver foil and fed into the combustion chamber. Carbon generated from the combusted sample is separated using gas chromatograph and used for carbon isotope analysis. Δ¹³C analysis was done using continuous-flow isotope ratio mass spectrometer (Delta V-Advantage, Thermo-Finnigan, Germany) interfaced with an elemental analyzer (CN1112, Carlo Erba, Italy).

Growth response of germplasm lines

The germplasm lines showed significant differences among all growth traits studied, both under control and at 50% moisture stress (Table 1). The germplasm line MD-O58 recorded the maximum plant height of 132.41cm and 120.44 cm at field capacity and 50% moisture stress, respectively, followed by MD-013 (109.96 and 99.50 cm), MD-111 (103.85 and 93.90 cm), S-268 (102.86 and 93.80 cm), and S-262 (96.67 and 87.86 cm), and the least was recorded in the germplasm line S-159 (45.56 and 41.00 cm). The total leaf area and number of leaves varied significantly (Table 1), with MD-058 showing the highest total leaf area (2645.38 and 2518.41 cm²) as well as the total number of leaves (536.37 and 494.00), while the lowest leaf area (1227.32 and 1162.46 cm²) and leaf number (313.20 and 196.02) were recorded in line S-159. The specific leaf area (SLA) varied significantly, with values ranging from 205.10 g cm^− 2 to the highest of 231.18 g cm^− 2 (in the lines S-159 and MD-013, respectively) under field capacity, and 173.80 g cm^− 2 and 206.17 g cm^− 2 (in S-159 and MD-058, respectively) under 50% moisture stress conditions.

Table 1

Growth attributes of germplasm lines grown at field capacity (FC) and 50% of moisture stress
Melia dubia Accession lines	Plant height (cm)		Total No. of leaves		Total leaf area (cm²)		SLA (g/ cm²)		Root Volume		Root: Shoot		Total dry matter (g)
Melia dubia Accession lines	FC	50% Stress	FC	50% Stress	FC	50% Stress	FC	50% Stress	FC	50% Stress	FC	50% Stress	FC	50% Stress
MD-058	132.41	120.44	536.37	494.00	2645.38	2518.41	227.76	189.91	119.22	108.80	0.31	0.48	133.59	129.94
MD-013	109.96	99.50	521.15	443.00	2272.51	2165.73	237.56	206.17	111.53	105.40	0.37	0.50	130.20	123.79
MD-111	103.85	93.90	482.56	438.20	2191.18	2048.64	231.18	190.22	109.81	104.00	0.39	0.46	117.54	111.01
S-268	102.86	93.80	495.78	464.00	2092.71	1906.43	236.61	200.14	101.64	95.80	0.37	0.47	109.30	104.10
S-262	96.67	87.76	502.11	428.40	1982.36	1884.99	232.85	201.91	91.45	84.20	0.38	0.50	103.87	99.26
MD-117	95.32	87.60	482.48	445.80	1792.71	1614.38	213.91	177.56	89.16	82.40	0.47	0.64	96.67	91.10
MD-115	82.42	75.60	443.40	422.62	1586.59	1411.87	206.85	174.85	79.58	73.00	0.48	0.71	93.60	86.21
S-270	78.89	72.60	388.67	370.60	1512.32	1411.09	209.19	187.17	68.42	59.80	0.47	0.66	87.24	80.60
S-069	58.49	53.33	353.43	350.00	1236.89	1187.17	207.45	174.74	87.08	84.03	0.43	0.61	79.20	76.30
S-159	45.56	41.00	313.20	196.02	1227.32	1162.46	205.10	173.80	58.25	50.20	0.42	0.63	59.49	55.39
CD @5%	15.16	13.15	146.00	151.19	526.14	514.58	13.10	16.43	35.72	34.14	NS	0.16	29.96	30.18
SE(m)	5.92	4.58	50.89	52.70	182.32	179.37	4.41	5.53	12.86	11.90	0.05	0.05	10.44	10.52

Root volume of the germplasm lines varied significantly, with the germplasm line MD-058 (119.22 and 108.80 cm³) recording the highest, followed by MD-013 (111.53 and 105.40 cm³), MD-111 (109.81 and 104.00 cm³), and S-69 (107.08 and 103.40 cm³). At both moisture regimes, S-159 had the lowest root volume (58.25 and 50.20 cm³). The root-to-shoot ratio varied significantly from 0.31 to 0.71 in MD-058 to 0.51 to 0.71in MD-115. Four out of five germplasm lines that recorded the highest biomass showed the lowest root-to-shoot ratios; similarly, four out of five lines that recorded lower biomass showed higher root-to-shoot ratios (Table 1). The total dry matter (TDM) of the plant varied significantly among the germplasm lines (Table 1). MD-058 had the highest dry matter at both moisture regimes (133.59 and 129.94 g plant^− 1), followed by MD-013 (130.20 and 123.79 g plant^− 1), and MD-111 (117.54 and 111.01 g plant^− 1), with S-159 having the lowest (59.49 and 55.39 g plant^− 1). The growth trends of ten selected germplasm lines were identical to those found in field circumstances.

The leaf is considered to be the most active plant part through which soil, plant, and environment interact (Lawson and Blatt 2014; Lopez et al. 1994) in co-ordinating energy, CO₂, and moisture (Atwell et al. 1999; Tuzet et al. 2003). Therefore, the leaf's role is considered critical in growth regulation (Warren and Adams 2000), especially under moisture stress conditions (Craufurd et al. 1993). The germplasm lines that recorded higher leaf area (for example, MD-058, MD-013, and MD-111) have also recorded higher growth, while the opposite is true in lines (S-159 and S-69) with poor growth (Table 1). More leaves in germplasm lines with higher growth are possible because of their higher plant height, which not only accommodates more leaves but also provides the necessary sink to utilize the assimilated carbon (Smith et al. 2018). The germplasm lines with the highest growth have also recorded higher SLA, indicating higher leaf thickness. With an increase in leaf thickness, the efficiency of carboxylation and photochemical reactions during photosynthesis are reported to increase (Evans and Poorter, 2001); therefore, photosynthesis increases. Such a response is observed in the germplasm lines with the highest growth, even under moisture-stress conditions. They recorded higher photosynthetic rates, and the saturation of photosynthesis was found to occur at higher light intensities (Fig. 3). Therefore, the germplasm lines that performed best at 50% moisture stress in this study, which are the same lines that showed similar performance under field conditions after four years of growth, can be considered as drought tolerant. And this can be attributed to their photosynthetic ability under moisture stress.

Apart from the leaf, the other most important morphological plant trait is the root system, which is crucial in sustaining growth under moisture stress. Apart from maintaining tissue water status, roots are also critical for maintaining the turgidity of cells, especially the guard cells that regulate the stomatal movement (Buckley 2019) which is critical in photosynthesis and transpiration. The partition of total biomass towards root growth in a plant depends on the growing conditions (Davies et al. 2011) and on the species (Luo et al. 2020; Eziz et al. 2017), according to the theory of optimum resource allocation (West et al. 1997). Among the germplasm lines, those with better growth allocate more biomass towards root growth (MD-058, MD-013, and MD-111) compared to poor-performing lines. This is evident from the root-shoot ratios of the germplasm lines (Table 1). Root volume also showed a similar trend among the germplasm lines. These trends suggest that resource generation and its utilization efficiency increase the “fitness” of plants and help to cope with moisture stress (Zhao et al. 2020; Wullschleger 1993). This type of adaptive response has already been reported in other species (Attia et al. 2015; McDowell et al. 2008) and has been found helpful in overcoming carbon starvation in situations where the drought is not intense enough to cause hydraulic failure but severe enough to drain the reserve carbon (Attia et al. 2015; McDowell et al. 2008). One of the many approaches to enduring moisture stress is to optimize resource generation and efficient utilization.

Photosynthesis response

The germplasm lines showed significant variations in carbon assimilation at two moisture regimes when measured using the gas exchange method (Table 2). MD-058 (18.17 µmole m^− 2s^− 1) and MD-013 (17.03 µmole m^− 2s^− 1) recorded the highest assimilation rate when the soil moisture was at field capacity and decreased by about 20.23 and 28.20%, respectively, when soil moisture was reduced by 50%. These two germplasm lines showed the least reduction in photosynthesis when exposed to moisture stress. In general, the reduction due to moisture stress is relatively less among the best-performing lines compared to poor growth lines (Table 1). For instance, the lines S-159 recorded the lowest photosynthesis (12.20 µmol CO₂ m^− 2s^− 1), and the reduction due to stress was 50.25%. Five germplasm lines selected as poor performers from their four years of field growth have recorded lower carbon assimilation rates (Table 2). The cumulative carbon assimilation rates among the germplasm lines revealed similar trends when measured using the stable carbon isotope (Δ¹³C) method. The germplasm line MD-058 (20.57‰) recorded the maximum Δ¹³C value, while S-270, S-69, and S-159 (Table 2) recorded the lowest. Similarly, NAR, which is again an indicator of cumulative carbon assimilation rate based on actual biomass production, also showed similar trends of carbon assimilation as well as growth (Table 2). The carbon assimilation rates showed similar trends in all three methods. Interestingly, the germplasm lines showed a strong positive relationship between carbon assimilation rates and biomass (Fig. 1). In all germplasm lines, photosynthesis saturation occurred between 1000 and 1500 µmol m^− 2 s^− 1 (Fig. 2), excepting S-159, S-270, and MD-117, in which it occurred around 1000 µmol m^− 2 s^− 1. The difference between the two moisture regimes among all the germplasm lines at all the light intensities did not differ much; however, the maximum photosynthetic rates among the germplasm lines varied. Among the best-performing germplasm lines (MD-058, MD-013, and MD-111), photosynthesis ranged between 18–20 µmol CO₂ m^− 2 s^− 1 at FC, while in S-270, S-69, and S-159, which are the poor performing lines, the assimilation rates were in the range of 10–13 µmol CO₂ m^− 2 s^− 1. The assimilation rates decreased in all the lines at 50% moisture stress (Table 2). The CO₂ response curves also showed similar trends (Fig. 3).

Table 2

Photosynthesis response of germplasm lines grown at field capacity (FC) and at 50% moisture stress. Values given in the parenthesis is the percent difference at 50% moisture stress over FC.
Melia dubia Accession lines	A (µmol CO₂ m^− 2 s^− 1)		Δ¹³C (‰)		NAR (mg/cm²/d)
Melia dubia Accession lines	FC	50% stress	FC	50% stress	FC	50% stress
MD-058	18.17	14.47 (20.23)	20.57	20.26 (1.50)	0.23	0.16 (30.43)
MD-013	16.53	11.97 (28.20)	20.31	20.02 (1.43)	0.19	0.14 (30.43)
MD-111	17.03	12.90 (24.26)	19.99	19.53 (2.30)	0.18	0.13 (27.77)
S-268	15.83	10.00 (36.83)	19.94	19.63 (1.55)	0.17	0.13 (23.52)
S-262	15.80	9.87 (62.46)	19.45	18.42 (5.29)	0.17	0.12 (29.41)
MD-117	15.63	9.53 (39.00)	20.25	19.07 (5.82)	0.16	0.10 (37.50)
MD-115	15.50	9.33 (39.81)	19.66	18.85 (4.12)	0.15	0.10 (33.33)
S-270	15.47	8.13 (47.45)	19.09	18.59 (2.61)	0.14	0.09 (35.71)
S-69	14.60	8.03 (45.00)	19.32	18.69 (3.26)	0.11	0.07 (36.36)
S-159	12.20	6.07 (50.25)	19.16	18.26 (4.69)	0.10	0.07 (30.0)
CD @5%	3.12	0.95	NS	0.62	0.04	0.04
SE(m)	1.05	0.32	0.49	0.21	0.01	0.02

The germplasm lines (MD-058, MD-111, and MD-013) with higher total dry matter (Table 1) also show higher photosynthesis at FC and moisture stress (Table 2). Under moisture stress conditions, the photosynthetic rates in these germplasm lines are as high as those of the germplasm lines S-159, S-69, and S-270 (which recorded the lowest growth rates) at FC. Since photosynthesis is the only physiological process that produces the substrate necessary for growth, enhancing this process, even in small proportions, is shown to profoundly help plant growth (Zhu et al. 2007), especially under moisture-stress conditions. The above results suggest that plants tend to increase their fitness to endure drought by maintaining higher carbon assimilation rates under moisture stress. The carbon assimilated is not only useful for growth but also for regulating the osmotic potential of the cells (Wang and Stutte 1992), osmotic homeostasis, stabilization of membranes, protein synthesis, and gene expression, as well as for carbohydrate and lipid metabolism (Rosa et al. 2009; Hoekstra et al. 2001). Hence, sustaining photosynthesis, even marginally, can substantially contribute to enduring moisture stress (Attia et al. 2015).

The transpiration rate in terms of CWT at two moisture levels among the germplasm lines was significantly different (Table 3). It was highest in MD-058 (14.49 lt plant^− 1), followed by MD-013 (13.77 lt plant^− 1) and MD-111 (13.59 lt plant^− 1), while it was least in S-159 (11.50 lt plant^− 1). The germplasm line MD-058 that recorded the highest transpiration rate when soil moisture was at field capacity showed the least reduction (19.52%) when exposed to 50% moisture stress, while the highest reduction of 38.96% was noticed in S-159 at 50% stress, which recorded the lowest CWT (7.02 lts plant^− 1) at field capacity. The daily transpiration rates (MTR lt plant^− 1) also showed similar trends. The drought susceptibility index was lowest in MD-058 (7.11%) and MD-013 (7.31%), while it was highest in S-159 (15.68%). The germplasm lines with higher carbon assimilation rates and growth rates (MD-058, MD-111, and MD-013) also show higher stomatal conductance and transpiration (Table 3). They also displayed a higher root volume (Table 1), suggesting that the moisture needed to maintain the higher transpiration loss is being augmented by roots through deeper mining of the soil. These germplasm lines have also shown better drought tolerance, which is evident from the lower drought susceptibility index (Table 3). Higher stomatal conductance is indispensable for maintaining higher photosynthetic rates (Farquhar 1982; Farquhar and Richards 1984; Farquhar et al. 1989). Most plant species are to maintain a steady leaf water potential (thereby stomatal conductance and transpiration) irrespective of soil moisture status, which is referred to as isohydric stomatal response (Sade et al. 2012). However, some plants and plant species maintain higher leaf water potential and thereby higher stomatal conductance despite decreasing soil moisture, which is referred to as anisohydric stomatal behaviour (Sade et al. 2012; Sala et al. 2010; McDowell et al. 2008; Martínez-Vilalta & Garcia-Forner 2017; Franks et al. 2007). Plants with anisohydric stomatal responses have also demonstrated higher photosynthetic rates and drought tolerance (Sade et al. 2012). Therefore, it may not be too hypothetical to believe that the M. dubia germplasm lines that grow better at 50% moisture have anisohydric stomatal behaviour. Such stomatal behaviour is reported among individual plants within a species (Sade et al. 2012) and in M. dubia (Tolia et al. 2019).

Table 3

Transpiration rates in terms of cumulative water transpired (CWT) of germplasm lines grown at FC and 50% moisture stress. Values indicated in the parenthesis are the percent difference at 50% moisture stress over FC.
M. dubia Accession lines	Stomatal Conductance (Gs) (mmol H₂O m^− 2 s^− 1)		CWT (lts/plant)		MTR (lts plant^− 1)		Drought Susceptibility index (%)
M. dubia Accession lines	FC	50% stress	FC	50% stress	FC	50%stress	Drought Susceptibility index (%)
MD-058	361.00	190.00 (20.36)	14.49	11.66 (19.53)	0.35	0.28 (20.00)	7.11
MD-013	320.00	157.00 (28.19)	13.77	10.80 (24.83)	0.33	0.26 (21.21)	7.31
MD-111	359.67	159.67 (24.25)	13.59	10.35 (23.84)	0.33	0.25 (24.42)	9.83
S-268	313.67	156.33 (36.83)	12.00	8.72 (27.33)	0.29	0.21 (27.58)	9.39
S-262	310.67	131.67 (37.53)	12.27	8.83 (28.03)	0.29	0.21 (27.58)	8.95
MD-117	306.00	99.33 (39.03)	12.45	8.51 (31.64)	0.30	0.20 (33.33)	10.02
MD-115	300.00	92.00 (39.81)	13.09	8.40 (35.82)	0.32	0.20 (37.50)	10.20
S-270	245.67	92.00 (47.45)	13.49	8.60 (36.24)	0.32	0.20 (37.50)	12.91
S-69	233.33	85.33 (45.00)	12.35	7.77 (58.94)	0.32	0.18 (43.75)	13.71
S-159	204.33	70.00 (50.25)	11.50	7.02 (38.95)	0.28	0.17 (39.28)	15.68
CD @5%	14.74	14.32	0.98	1.68	0.028	0.043	1.18
SE(m)	4.96	4.82	0.33	0.56	0.009	0.014	0.40

The stomatal conductance was highest in MD-058 under both moisture regimes (361 and 290 mmol H₂O m^− 2 s^− 1), while it was lowest in S-159 (204.33 and 170 mmol H₂O m^− 2 s^− 1). The germplasm lines with a higher transpiration rate also showed higher stomatal conductance (Table 3) and photosynthetic rate. A strong relationship between the stomatal conductance, the photosynthetic rate, and transpiration is noticed at both moisture regimes. The above results are subject to further scrutiny using the stable carbon isotope discrimination approach (Table 2). This approach is considered a more reliable surrogate for stomatal behaviour on a cumulative scale (Farquhar, 1982). Due to moisture stress, stomatal conductance decreases, as does CO₂ diffusion. With an increase in stomatal resistance, the proportion of the ¹³C isotope will increase compared to the ¹²C isotope because it is heavier than ¹²C (Griffiths 1993). Such discrimination against ¹³C also occurs during the entire process of carbon fixation (dissolution and carboxylation processes). However, the discrimination between ¹³C and ¹²C decreases under stress because of the lower substrate availability (CO₂). As a result, the proportion of ¹³C increases in the carbon assimilated during stress. In other words, the ratio of Δ¹³C/Δ¹²C values in the biomass increases in plants under moisture stress. Thus, the lower differences in Δ¹³C/Δ¹²C between the stressed and unstressed plants among the better-performing germplasm lines also recorded higher growth, suggesting higher stomatal conductance under moisture stress (Table 2).

The WUE varied considerably among the germplasm lines under both moisture regimes. Germplasm line MD-058 recorded the highest water use efficiency (0.059 g l^− 1 day^− 1 and 0.042 g l^− 1 day^− 1) at FC and at 50% moisture stress, respectively (Table 4), while it was lowest in S-159 at FC (0.024 g l^− 1 day^− 1) as well as at 50% less than FC (0.015g l^− 1 day^− 1). The germplasm lines that recorded higher WUE also recorded the highest biomass, and vice versa (Table 1). Light and carbon dioxide are two critical environmental factors that directly influence the process of photosynthesis, as elucidated by the light and CO₂ response curves (Figs. 2 and 3). The carbon assimilation rate at saturating light intensities is considered to be a reflection of chloroplast capacity for carbon assimilation. A lesser reduction in the initial slope of the light response curves of MD-058, MD-013, and MD-111 (Fig. 2) under moisture stress is an indication of better quantum efficiency (Farquhar and Richards 1984), while higher initial slopes of CO₂ response curves (Fig. 3) are an indication of chloroplast efficiency to utilize incident light in the carboxylation process (Terashima et al. 2005). Thus, higher carbon assimilation rates in these lines under moisture-stress conditions are clearly evident from the results. The light saturation of photosynthesis around 1500–1750 µmol m^− 2s^− 1 in all the lines indicates higher light use efficiency in M. dubia, which is a tropical, sun-loving tree species. The higher photosynthetic rates in general of M. dubia observed indicate the higher carbon assimilation capability of this species and hence its growth.

Table 4

Water use efficiency (WUE) of germplasm lines grown at FC and 50% moisture stress. Values in the parenthesis are percentile difference at 50% moisture stress over FC.
M. dubia Accession lines	WUE (g L^− 1 day^− 1)
M. dubia Accession lines	FC	50% moisture stress
MD-058	0.059	0.042 (28.81)
MD-013	0.049	0.038 (22.44)
MD-111	0.054	0.034 (37.03)
S-268	0.043	0.035 (18.60)
S-262	0.048	0.029 (39.58)
MD-117	0.039	0.024 (38.46)
MD-115	0.037	0.023 (37.83)
S-270	0.039	0.023 (37.83)
S-69	0.033	0.023 (30.30)
S-159	0.024	0.015 (37.50)
CD @5%	0.012	0.006
SE(m)	0.004	0.002

Germplasm lines with the highest and lowest growth when grown under rainfed conditions (that represent the growth under intermittent moisture stress conditions) showed similar growth responses when exposed to 50% moisture stress under controlled conditions in the seedlings. Germplasm lines with higher growth also exhibited a higher photosynthetic rate as well as higher moisture stress tolerance, suggesting that higher resource generation increases plant fitness to sustain moisture stress. Factors such as photosynthetic surface area, photosynthetic rate, and distribution and utilization of the assimilated carbon under moisture stress observed in these germplasm lines further substantiate the role of photosynthesis in drought tolerance. The significance of their resource-generating ability to sustain moisture stress is evident in their photosynthetic and growth responses. The growth of germplasm lines (as trees) noticed in the field after four years, which represent the performance under intermittent moisture stress, and growth observed under controlled moisture stress conditions, suggests that the photosynthetic capacity of a plant can be a reliable trait in identifying drought tolerance, even at the seedling stage. Thus, photosynthesis being the only resource-generating process in a plant, it will have a strong bearing on growth in general, and certainly under moisture stress conditions.

Acknowledgement: We acknowledge the INSPIRE Fellowship (IF 140724) provided by DST, New Delhi, India, for the doctoral program of Loushambam Romeechand Singh. We sincerely thank Dr. M.S. Sheshshayee for providing the stable carbon isotope analysis facility. We thank the Karnataka State Forest Department for granting permission to carry out this study in their research station at Hoskote. Our sincere thanks to Prof. M. S. Sheshshayee, Professor, Department of Crop Physiology, UAS, Bangalore, for his help in stable carbon isotope analysis.

Funding: Nil

Competing interests: The authors have no conflicts of interest to declare relevant to this article's content.

Availability of data and material: All the data is presented in the article and as online resource.

Code availability: Not applicable

Authors' contributions (optional: please review the submission guidelines from the journal whether statements are mandatory)

Loushambam Romeechand Singh – Field work, data collection, analysis and compilation of data and assistance in manuscript preparation

Devakumar A. S. – Conceptualization of the study, planning, executing, facilitating lab and field experiments and manuscript preparation.

Ethics approval: Not applicable

Consent to participate: Consent is obtained from all the participating researchers.

Consent for publication: Consent from all participating researchers has been obtained for publishing results.

Key Message: Sustaining photosynthesis under moisture stress induces drought tolerance in germplasm lines of M. dubia, which performed better under drought conditions.

Attia Z, Jean-Christophe Domec, Ram Oren, Way DA, Moshelion M (2015) Growth and physiological responses of isohydric and anisohydric poplars to drought. J Expt Bot 66 (14): 4373–4381. doi: 10.1093/jxb/erv195.
Atwell S, Huang YS, Vilhjálmsson BJ (1999) Genome-wide association study of 107 phenotypes in Arabidopsis thaliana inbred lines. Nature 465: 627–631. doi: 10.1038/nature08800.
Betts R, Cox P, Collins M, et al. (2004) The role of ecosystem-atmosphere interactions in simulated Amazonian precipitation decrease and forest dieback under global climate warming. Theor Appl Climatol 78,157–175 https://doi.org/10.1007/s00704-004-0050-y
Bre´da N, Huc R, Granier A, Dreyer E (2006) Temperate forest trees and stands under severe drought: a review of ecophysiological responses, adaptation processes and long-term consequences. Ann For Sci 63:625–644. DOI: 10.1051/forest:2006042
Brienen RJW, Lebrija TE, Vanbreugel M, Perez-Garcia EA, Bongers F, Meave JA, Marti´Nez M (2011) Stable carbon isotopes in tree rings indicate improved water use efficiency and drought responses of a tropical dry forest tree species. Trees 25:103–113
Buckley TN (2019) How do stomata respond to water status? New phytologist.
Craufurd PQ, Flowe, DJ, Peacock JM (1993) Effect of heat and drought stress on sorghum (Sorghum bicolor) panicle development and leaf appearance. Expt Agric 29: 61-76. DOI:10.1017/S0014479700020421
Davies WJ, Zhang J, Yang J, Dodd IC (2011) Novel crop science to improve yield and resource use efficiency in water-limited agriculture. J Agric Sci 149: 123–131. https://doi.org/10.1017/S0021859610001115
Devakumar AS, Gawai PP, Sathik MBM, James J (1999) Drought alters the canopy architecture and micro-climate of Hevea brasiliensis. Trees 13: 161-167. https://doi.org/10.1007/PL00009747
Döös BR (2002) Population growth and loss of arable land, Global Environmental Change,12 (4): 303-311. https://doi.org/10.1016/S0959-3780(02)00043-2.
Evans JR, Poorter H (2001) Photosynthetic acclimation of plants to growth irradiance: the relative importance of specific leaf area and nitrogen partitioning in maximizing carbon gain. Plant Cell Environ 24: 55–767. https://doi.org/10.1046/j.1365-3040.2001.00724.x
Eziz A, Yan Z, Tian D, Han W, Tang Z, Fang J (2017) Drought effect on plant biomass allocation: A meta‐analysis. Ecol Evol https://doi.org/10.1002/ece3.3630
Farquhar GD (1982) On the relationship between carbon isotope discrimination and the intercellular CO₂ concentration in leaves. Aust J Plant Physiol 9: 121-131. DOI:10.1071/PP9820121
Farquhar GD, Ehleringer JR, Hubick KT (1989) Carbon isotope discrimination and photosynthesis. Annu Rev Plant Physiol Plant Mol Biol., 40: 503-537. https://doi.org/10.1146/annurev.pp.40.060189.002443
Farquhar GD, Richards RA (1984) Isotopic composition of plant carbon correlates with water use efficiency of wheat cultivars. Aust J Plant Physiol, 11: 539-552. DOI:10.1071/PP9840539
Fischer RA, Maurer R (1978) Drought resistance in spring wheat cultivars. Aust J Agric Res 29: 897–912
Francey RJ, Farquhar GD (1982) An explanation of ¹³C/¹²C variations in tree rings. Nature 297: 28–31
Franks PJ, Paul L, Drake, Ray H, Froend (2007) Anisohydric but isohydrodynamic: seasonally constant plant water potential gradient explained by a stomatal control mechanism incorporating variable plant hydraulic conductance. Plant Cell Environ 30:19–30 doi: 10.1111/j.1365-3040.2006.01600.x
Gore A (2022) Land degradation and desertification- Action points for India from UNCCD’s Global land outlook report 2022. Land Degradation and Desertification – Action Points for India from UNCCD’s Global Land Outlook Report 2022 - WOTR
Griffiths H (1993). Carbon isotope discrimination. In: Hall, D.O., Scurlock, J.M.O., Bolhàr-Nordenkampf, H.R., Leegood, R.C., Long, S.P. (eds) Photosynthesis and Production in a Changing Environment. Springer, Dordrecht. https://doi.org/10.1007/978-94-011-1566-7_11
Hoekstra FA, Golovina EA, Buitink J (2001) Mechanisms of plant desiccation tolerance. Trends in plant science 6(9):431-438 https://doi.org/10.1016/S1360-1385(01)02052-0
Hubick KT, Farquhar GD (1989) Carbon isotope discrimination and ratio of carbon gained to water lost in barley cultivars. Plant Cell Environ 12: 795-804. https://doi.org/10.1111/j.1365-3040.1989.tb01641.x
IPCC (2007) Climate Change 2007 Mitigation of Climate Change. Working Group III contribution to the Intergovernmental Panel on Climate Change, Fourth Assessment Report. Bangkok, Thailand. https://www.ipcc.ch/pdf/assessment report/ar4/wg3/ar4_wg3_full_report.pdf (Accessed 28 January 2008)
Lawson T, Blatt MR, (2014) Stomatal Size, Speed, and Responsiveness Impact on Photosynthesis and Water Use Efﬁciency. Plant physiol 164:1556-1570. doi: 10.1104/pp.114.237107. Epub 2014 Feb 27.
Lopez FB, Chauhan YS, Johansen C (1994) Effects of Timing of Drought Stress on Phenology, Yield and Yield Components of Short-duration Pigeonpea. J Agronomy & Crop Science 178: 1-7
Luo YZ, LiG, YanG, Liu H, Turner NC(2020) Morphological Features and Biomass Partitioning of Lucerne Plants (Medicago sativa L.) Subjected to Water Stress. Agron 10(3): 322 https://doi.org/10.3390/agronomy10030322
Maire V, Gross N, Hill D, Martin R, Wirth IJ, Soussana JF (2013) Disentangling coordination among functional traits using an individual-centered model; impact on plant performance at intra and inter-speciﬁc levels. PLOSOne 8(10): e77372.
Manzoni S, Vico G, Katul G, Palmroth S, Porporato A (2014) Optimal plant water-use strategies under stochastic rainfall. Water Resour Res 50:5379–5394
Martínez-Vilalta J, Garcia-Forner N (2017) Water potential regulation, stomatal behaviour and hydraulic transport under drought: deconstructing the iso/anisohydric concept Plant, Cell and Environment (2017) 40, 962–976 doi: 10.1111/pce.12846
McDowell N, Pockman WT, Allen CD, Breshears DD, Cobb N, Kolb T, Plaut J, Sperry J, West A, Williams DG,Yepez EA (2008) Mechanisms of plant survival and mortality during drought: why do some plants survive while others succumb to drought? New Phytol 178:719-739
NOAA (2022) Greenhouse gases continued to increase rapidly in 2022 | National Oceanic and Atmospheric Administration (noaa.gov)
Osório J, Pereira JS (1994) Genotypic differences in water-use efficiency and 13C discrimination in Eucalyptus globulus. Tree Physiol 14: 871–882
Parthiban KT, Bharathi AK, Seenivasan R, Kamala K, Rao MG (2009) Integrating Melia dubia in agroforestry farms as an alternate pulpwood species. Asia-Pacific Agroforestry Network 34: 3-4
Peel MC, Finlayson BL, Mcmahon C (2007) Updated World Map of the Koppen-Geiger Climate Classification. Hydrol Earth Syst Sci 11:1633–1644DOI:10.5194/hess-11-1633-2007
Rosa M, Prado C, Podazza G, Interdonato R, González JA, Hilal M, Fernando E. Prado (2009) Soluble sugars. Plant Signal Behav 4: (5) 388-393, DOI: 10.4161/psb.4.5.8294
Rowell K, Dettman DL, Dietz R (2009) Nitrogen isotopes in otoliths reconstruct ancient trophic position. Environ Bio Fish 89: 415–425.
Sade N, Gebremedhin A, Moshelion M (2012) Risk-taking plants;Anisohydric behavior as a stress-resistance trait. Plant Signal Behav. 7(7): 767–770. doi: 10.4161/psb.20505
Sala A, Piper H, Hoch G (2010) Physiological mechanisms of drought‐induced tree mortality are far from being resolved. New Phytol https://doi.org/10.1111/j.1469-8137.2009.03167.x
Seager MF, Ting IM, Held Y, Kushnir J, Lu G, Vecchi HP, Huang N, Harnik A, Leetmaa NC, Lau C, Li J, Velez N (2007) Model Projections of an Imminent Transition to a More Arid Climate in Southwestern North America. Science 316: 1181 – 1184 DOI:10.1126/science.1139601
Singh JS, Joshi M (1979) Ecology of the Semi-Arid Regions of India with Emphasis on Land-Use in WALKER BH (ed) Developments in Agricultural and Managed Forest Ecology, Elsevier, Volume 7, Pp 243-275 https://doi.org/10.1016/B978-0-444-41759-6.50012-8.
Smith MR, Rao IM, Merchant A (2018) Source-Sink Relationships in Crop Plants and Their Influence on Yield Development and Nutritional Quality.Front Plant Sci 20 December 2018 https://doi.org/10.3389/fpls.2018.01889
Stern N (2007) The Economics of Climate Change: The Stern Review. Cambridge University Press, Cambridge, UK
Tardieu F (2013) Plant response to environmental conditions: assessing potential production, water demand, and negative effects of water deficit. Front Physiol 4(17): 1-11
Terashima I, Araya, T, Miyazawa SI , Sone K, Yano S ( 2005 ) Construction and maintenance of the optimal photosynthetic systems of the leaf, herbaceous plant and tree: an eco-developmental treatise. Ann Bot 95: 507 – 519
Tolia N, Devakumar AS, Sheshshayee MS, Kamblimath K (2019) Growth performance of six multipurpose tree species based on the carbon assimilation capacity: a functional approach. Agroforest System 93(3): 1031-1043
Tuzet A, Perrier A, Leuning R (2003) A coupled model of stomatal conductance, photosynthesis and transpiration. Plant Cell Environ 26: 1097–1116
Udayakumar M, Rao RCN, Wright GC, Ramaswamy GC, Roy S, Gangadhar GC, Aftab HIS (1998) Measurement of transpiration efficiency in field condition. J Plant Physiol Biochem 1: 69-75
Venkateshwarulu B (2019) Rainfed Agriculture in India: Issues in technology development and transfer. https://krishi.icar.gov.in/jspui/bitstream/123456789/32972/1/BV.pdf accessed on 26/11/2020
Wang Z, Gary W, Stutte (1992) The Role of Carbohydrates in Active Osmotic Adjustment in Apple under Water Stress. J Am Soc Hortic 117(5):816-823. doi:10.1093/jxb/erv195
Warren CR, Adams MA (2000) Trade-offs between the persistence of foliage and productivity in two Pinus species. Oecologia 124: 487–494
Warrier RR (2012) Melia dubia Cav. Institute of forest genetics and tree breeding. Coimbatore
West GB, Brown JH, Enquist BJ (1997) A general model for the origin of allometric scaling laws in biology. Science 276:122– 126. https://doi.org/10.1126/science.276.5309.122
Winter SR, Musick JT, Porter KB (1988) Evaluation of screening techniques for breeding drought-resistant winter wheat. Crop Sci 28:512–516
Wullschleger SD (1993) Biochemical Limitations to Carbon Assimilation in C3 Plants—A Retrospective Analysis of the A/Ci Curves from 109 Species. J Expt Bot 44(262):907-920
Yang Y, Sarah E, Rebecca H, Hernandez R, Fargione J, Grodsky SM, Tilman D, Zhu YG, Luo Y, Smith TM, Jungers JM, Yang M, Chen WQ (2020) Restoring Abandoned Farmland to Mitigate Climate Change on a Full Earth. One Earth 3(2):176-186 https://doi.org/10.1016/j.oneear.2020.07.019.
Zhao W, Liu L, Shen Q, Yang J, Han X, Tian F, Wu J (2020) Effects of Water Stress on Photosynthesis, Yield, and Water Use Efficiency in Winter Wheat. Water12: 2127 doi:10.3390/w12082127
Zhu XG, de Sturler E, Long SP (2007) Optimizing the distribution of resources between enzymes of carbon metabolism can dramatically increase photosynthetic rate: a numerical simulation using an evolutionary algorithm. Plant Physiol 145: 513–526
Zobel B, Talbert J (1984) Applied forest Tree improvement. The Black burn press, USA

Supplementarytable.docx

Resource generation is critical to sustain moisture stress in Melia dubia Cav.

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Abstract

Figures

Introduction

Materials and methods

Gravimetric approach for assessing the water use efficiency (WUE)

Growth assessment

Photosynthesis measurement

Results and Discussion

Growth response of germplasm lines

Photosynthesis response

Conclusion

Declarations

References

Supplementary Files

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