Variation in biochemical, physiological and ecophysiological traits among the teak (Tectona grandis Linn. f) seed sources of India

Teak being an iconic timber species the studies on its physiological and biochemical traits are very limited in India and worldwide. As a result, the current study aimed to assess biochemical parameters such as chlorophyll a, chlorophyll b, total chlorophyll, carotenoids, chlorophyll ab ratio, proline content, and peroxidase activity, along with physiological parameters such as Chlorophyll stability index, relative water content, and leaf area, as well as ecophysiological traits such as net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), transpiration rate (Tr), Leaf temperature, intrinsic water-use efficiency (iWUE), instantaneous water use efficiency and intrinsic carboxylation efficiency of thirty teak seed sources collected from different states of India. FCRITK 19, FCRITK 21, FCRITK 25, FCRITK 29, and FCRITK 05 were reported to have a greater photosynthetic rate (> 17 µmol m−2 s−1) coupled with a relative water content of more than 50% and a chlorophyll stability index of more than 60%, which could be used in a future genetic improvement programme. Correlation analysis indicated that water use efficiency was found to be strongly but negatively correlated with transpiration rate (−0.601) and stomatal conductance (−0.910). The proline content had a substantial positive correlation with the chlorophyll stability index (0.890), signifying that they are associated with abiotic stress conditions. Cluster analysis was attempted to discriminate the sources based on biochemical, physiological and ecophysiological traits. Eleven sources (FCRITK 25, FCRITK 27, FCRITK 29, FCRITK 14, FCRITK 30, FCRITK 16, FCRITK 05, FCRITK 13, FCRITK 02, FCRITK 17 and FCRITK 15) exhibited superior performance compared to rest of the sources.

for maximizing productivity, especially because teak yields and quality vary a lot depending on planting site conditions 4,10 . The photosynthetic features are the key quantifiable indicators of plant growth. Information on variation in photosynthetic parameters and their relationship with growth traits helps us understand underlying processes and responses, and will be useful in tree improvement programs 11 .
Plant growth and survival will be harmed more severely and frequently as a result of climate change. The ability of a plant to cope with stress and recuperate determines its ability to modify growth and development under harsh situations. Growing periods with water scarcity can lead to decreased rates of height and diameter growth, reduced resistance to biotic and abiotic factors and changes in the timing and rate of physiological processes 12 . Teak plantations, like many other tropical plant species, are subjected to sustained periods of drought stress. As a result, it was expected that when plants were exposed to drought stress, a significant number of simultaneous changes in morphological, physiological, and biochemical responses would occur and that these changes would improve the plant's ability to survive and proliferate during drought times 13 . Teak being a widely adopted species the reports on its physiological and biochemical studies are very limited in India and worldwide. In 2013, the United Nations Climate Change Conference (UNFCCC) adopted seven agreements on forests, reiterating the importance of forests in reducing greenhouse gas emissions from industry. Carbon sequestration in terrestrial ecosystems can help humans adapt to present and future environmental changes by reducing the pace at which greenhouse gases accumulate in the atmosphere 14 . It is found that Teak plantations have a high potential for carbon sequestration, depending on the species, plantation techniques, and agronomical approaches used in maintenance and aftercare. Teak plants also aid in climate change mitigation by absorbing a variety of green gases, converting and fixing them into biomass, and returning oxygen to the environment. Aside from that, they have a lot of potential for enriching the forest floor to help with growth and biomass 15 .
Briggs and Shantz 16 created the concept of water use efficiency (WUE) 100 years ago, demonstrating a link between plant yield and water use. They used the phrase "water use efficiency" to describe how much biomass a plant produces per unit of water consumed. The ability of trees to exploit water and nutrient resources plays a significant role in their adaptability to environmental changes. The ratio of net photosynthetic CO 2 assimilation (A) to stomatal conductance (gs) is used to calculate tree-level intrinsic water-use efficiency (iWUE). In terrestrial ecosystems, iWUE is a crucial component of water-carbon coupling and process management, as well as a mechanism for trees to adapt to changing conditions 17 . The iWUE of teak seed sources could aid in improving drought tolerance in teak during advanced generations breeding programmes. Despite this, no attempt has been made to evaluate the relationship between WUE and other quantitative variables that affect species growth and most of the previous studies focus only on teak sources from limited areas. Against this backdrop, the objective of this study is to screen teak sources for high photosynthetic efficiency based on ecophysiological and biochemical parameters, as well as to understand the role of iWUE in tree growth and to identify the best sources for plantation development in dry areas and future genetic improvement work.

Materials and methods
The experiment was conducted in one year old seed source evaluation trial established at Forest College and Research Institute, Mettupalayam (11 • 19′ N; 76 • 56′ E; 300 m above MSL) during 2021. The materials for the present study consisted of 30 teak seed sources (Table 1) collected from selected plus trees of eleven different states viz., Tamil Nadu, Tripura, Maharashtra, Odisha, Gujarat, Kerala, Karnataka, Andhra Pradesh, Madhya Pradesh, Chhattisgarh and Jharkhand with the help of respective forest department officials along with proper permissions. The assembled seed sources were established in the seed source evaluation trial using Randomized Block Design (RBD) with 8 plants per plot with three replications at an espacement of 4 m × 4 m. All analyses were performed following the relevant guidelines and regulations. Experimental Material. (a) The seeds were collected from a group of phenotypically superior trees (plus trees) which were selected based on the comparison tree method (b) The collected seeds were sown in raised beds with a medium of red soil, sand and farm yard manure in the ratio of 2:1:1. The beds were watered at regular intervals and maintained for six months. After 6 months the stumps which are above 3-4 cm thickness at the collar region are selected and transplanted into polybags containing a medium of red soil, sand and farm yard manure (FYM) in the ratio of 2:1:1. After a month of transplantation the seedlings were planted in the main field (c) All the seeds were sown simultaneously within a timespan of a week (d) No chemical treatments or fertilizers were provided at the nursery stage. At the time of planting each seedling was supplemented with 250 g of FYM, 25 g of vermicompost and 5 g of DAP (Di Ammonium Phosphate). The following data were collected from oneyear-old grown teak plants.
Ecophysiological traits. The gas exchange parameters including net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO 2 concentration (Ci), transpiration rate (Tr) and Leaf temperature were measured using a Li 6400 photosynthetic system (Li-Cor, Inc., Nebraska, USA). From each replication, fully mature and expanded leaves of each source were measured during the period of 10:00 to 12:00 h in the morning.
The intrinsic water-use efficiency (iWUE) is defined as the ratio of net photosynthesis and stomatal conductance, expressed in the units of µmol mol −118 . Instantaneous water use efficiency was estimated as the ratio of net photosynthetic rate to transpiration rate 19 . The ratio of net photosynthetic rate to the intercellular CO 2 concentration is termed as intrinsic carboxylation efficiency 20 .
Determination of leaf area. The leaf area is estimated by the linear method as per Montgomery 21 by using the following formula where K represents concentration/absorbance.
Peroxidase assay was performed at 25C in 3 ml of 60 mm phosphate buffer (pH 6.1) containing 16 mM guaiacol and 2 mM H202. Increase in absorbance was recorded at 470 nm with a Unicam SP 1700 Spectrophotometer. The reaction was linear for 30 min. G6PDH activity was assayed in 1 ml 60 mM Tris-HCl (pH 8.1) buffer with Leaf area cm 2 = K (0.836) x L x B Proline µg/g = Absorbance of Sample * K Value * Dilution Factor / Weight of Sample g Statistical analysis. One-way ANOVA was used to analyze the data of physiological and biochemical traits of different teak seed sources, and Duncan's multiple range test was used to compare treatment means. The IBM-SPSS analytical software programme version 20.0 (IBM Corporation, USA) was used to analyze the data. The clustering analysis was performed by the UPGMA method (Unweighted Pair Group Method with Arithmetic mean) employing Past 4.03 software 27 .
Water use efficiency parameters like instantaneous water use efficiency, intrinsic water use efficiency and intrinsic carboxylation efficiency were found to be significant among the teak seed sources. Instantaneous water use efficiency ranged between 14.29 ± 0.42 (FCRITK 20) to 5.07 ± 0.11 (FCRITK 15) µmol mmol −1 . Intrinsic water use efficiency varied from 110.36 ± 0.27 (FCRITK 28) to 16.49 ± 0.82 (FCRITK 08) µmol mol −1 and intrinsic Table 2. Variance analysis (ANOVA) of biochemical parameters, physiological parameters and ecophysiological traits among teak seed sources. **Highly significant difference at p < 0.001 level of probability, and ns-no significance.  (Table 6) of teak sources showed that the chlorophyll stability index had a positive significant correlation with proline content (0.890) whereas it had a significant negative correlation with leaf temperature (−0.580). Similarly proline content also had a significant negative correlation with leaf temperature (−0.588). Height and basal diameter revealed a substantial positive relationship (0.733). Stomatal conductance had a positive significant correlation with transpiration rate (0.553) and a significant negative correlation with water use efficiency (−0.910). Total chlorophyll (−0.371) and transpiration rate (−0.601) were found to have a negative significant correlation with water use efficiency.
The data on biochemical, physiological and ecophysiological traits were analyzed using hierarchical cluster analysis, by UPGMA method based on Euclidian distance for the thirty teak seed sources (Fig. 1). The sources were segregated into twelve clusters where the cluster XI had eight sources, clusters X and V had four sources each, cluster VII had three sources, clusters II, III and VIII had two sources each, whereas the remaining clusters have one source each.

Discussion
The impacts of stress on photosynthetic physiology, as well as photosynthetic responses to light intensity and CO 2 concentration, have been the focus of ecophysiological investigations on photosynthesis in forest trees till date 28 . The current study highlights the physiological and biochemical characteristics of teak seed sources from different locations. The objectives of this paper was to systematically measure photosynthetic gas exchange and chlorophyll parameters, correlate photosynthetic and biochemical characteristics with growth, and provide a strategy for rapid evaluation of teak seed sources in order to introduce, use, and improve teak resources in future breeding programmes. Table 3. Values of biochemical parameters among teak seed sources from different states of India. Values followed by the different letter of each group were significantly different at p < 0.05 level of probability.

Sources
Chlorophyll a (mg g −1 ) Chlorophyll b (mg g −1 ) Total chlorophyll (mg g −1 ) Carotenoids (mg g −1 ) Chlorophyll ab ratio  29 . Changes in the amount of chlorophyll may also be a part of adaptive reactions. The current study implied that chlorophyll a, chlorophyll b, total chlorophyll and chlorophyll ab ratio registered significantly comparable values among the sources. The decreased values could be due to photoinhibition 30 . Chlorophyll loss is both a negative and adaptive component of stress since it reduces light absorption and hence the risk of further damage to the photosynthetic mechanism 31 . Higher values were noted in FCRITK 06, FCRITK 05, FCRITK 02 and FCRITK 26. Carotenoids play an important role in photosynthesis and photoprotection. They are well known for their antioxidant properties, in addition to their structural responsibilities. Carotenoids are also important for the formation of the light-harvesting complex and the nonradiative dissipation of surplus energy 31 . Carotenoids ranged from 1.070 ± 0.05 to 0.516 ± 0.02 mg g −1 . Similar trends in chlorophylls and carotenoids were noticed in Teak 12,13,30,32,33 , Pungam 34 and Rubber 35 . Many studies [36][37][38] demonstrate that mild stress does not affect chlorophyll concentration. Proline accumulation acts as an osmotic regulator and a protective mechanism in plants under abiotic stress 39 . Free proline is reported to induce stress tolerance in a variety of plants through dehydration of protoplasm. The amount of proline present in the teak clones was between the ranges of 3.76 ± 0.07 to 1.17 ± 0.05 µg g −1 which indicated that the plants can withstand abiotic stress conditions. The results are in corroboration with Teak 12,13,30 , Rubber 35 and Populus 40 .
Peroxidase activity is an adaptive feature that helps to repair tissue metabolic damage by lowering the harmful quantities of H 2 O 2 generated during cell metabolism. Peroxidase protects against oxidative stress by converting H 2 O 2 to water and oxygen. The increase in peroxidase activity implies that the plant uses protective mechanisms (photo-protection) to cope with moisture stress. Increased peroxidase activity could suggest that the cell wall's mechanical characteristics have deteriorated 12 . The present study indicates that all the teak sources exhibited reduced peroxidase activity (0.048 ± 0.00 to 0.030 ± 0.00 min −1 mg −1 ) which might be due to reduced stress conditions. Similar trends have been noticed in teak 12,13 and Populus cathayana 41 . Table 4. Values of physiological parameters among teak seed sources from different states of India. Values followed by the different letter of each group were significantly different at p < 0.05 level of probability.

Sources
Chlorophyll stability index (%) Relative water content (%) Leaf Area (cm 2 )  Ecophysiological traits. The leaf is one of the most important organs in the plant system, and the plant's continuous development is dependent on its ability to persist. Physiologically, the leaf area represents the principal photosynthetic surface and supplies most of the photosynthates required by the plant components. As a result, estimating leaf area becomes an important aspect of growth analysis and is frequently used in physiological reasoning of agricultural productivity fluctuations. The leaf area of teak sources ranged from 3469.12 ± 120.49 to 1292.32 ± 19.90 cm 2 which revealed that all the sources exhibited higher leaf area than other findings in teak 30,43 . Photosynthesis is crucial for plant development and productivity. Plant photosynthesis is influenced not only by environmental conditions but also by the genetic traits of the plant. Photosynthetic activity is influenced by a complicated process of interaction between genetic and environmental factors in plants 44 . The photosynthetic rate was found to be higher in FCRITK 19, FCRITK 21, FCRITK 25, FCRITK 29 and FCRITK 05 indicating that these seed sources exhibit higher productivity in terms of biomass. Stomatal conductance was found to be in the higher range (0.87 to 0.14 mol m −2 s −1 ) which might be due to increased leaf temperature (38.80 to 37.15 °C) leading to increased photosynthesis 45 . Transpiration rate was found to be low among the teak seed sources which specifies that these sources can be utilized for plantation under drought-prone areas. Intercellular CO 2 concentration ranged between 475.0 ± 15.23 to 180.5 ± 6.49 µl l −1 indicating that higher intercellular CO 2 concentration associated with increased stomatal conductance affects the photosynthetic rate. Lower photosynthetic rate than the current investigation was documented in teak [11][12][13]33,42,46 , Similar noteworthy results were documented in Pawlonia tomentosa 47 , Eucalyptus species 48 , Tabebuia aurea 49 and Rubber 35 .
The WUE of plants could be used as a key criterion for developing drought-tolerant varieties 50 . Instantaneous water use efficiency was found to be higher among teak sources in the current investigation, indicating that these sources are better at diverting water for photosynthesis than transpiration. The intrinsic water use efficiency varied significantly in the present study. Higher iWUE values (FCRITK 28, FCRITK 29, FCRITK01 and FCRITK 22) imply that these teak sources are better at carbon assimilation, resulting in higher productivity under drought stress. Other such findings in eucalyptus clones [51][52][53] and teak 11,54 lend support to the present investigation.
Correlation analysis. Correlation analysis indicated that water use efficiency was significantly but negatively correlated with transpiration rate and stomatal conductance suggesting that the WUE may decrease when transpiration rate and stomatal conductance is high. Leaf temperature had a significant negative correlation with chlorophyll stability index and proline content. Chlorophyll stability index had a significant positive correlation with proline content indicating that they are directly related to abiotic stress conditions. Similar trends were noted in eucalyptus clones 53 . Significant correlations between growth traits and physiological parameters were observed in Salix species under normal conditions 55 and in Teak under drought conditions 13 . Under drought stress conditions, positive correlations between chlorophyll content, growth traits, and physiological parameters were also found in other species such as Alstonia macrophylla, Acacia auriculiformis, Artocarpus heterophyllus, Terminalia arjuna and Azadiracta indica 56 . Cluster analysis. Cluster analysis was attempted to discriminate the sources based on biochemical, physio-

Conclusion
Plants are capable of adapting to abiotic stress and phenotypic plasticity in response to it. As a result, the ability of plants to respond to stress, as well as their ability to recover from stress and resume normal metabolism, is crucial. As a result, knowing biochemical, physiological, and ecophysiological features is crucial for tackling global climate change-related challenges, such as drought. In this study, teak seed sources FCRITK 19, FCRITK 21, FCRITK 25, FCRITK 29, and FCRITK 05 were discovered to have a higher photosynthetic rate, as well as a relative water content of more than 50% and a chlorophyll stability index of more than 60%, and could be used in a future genetic improvement programme. Continued research is essential on the correlation of these traits with genetic mechanisms, including the identification of potential genes linked to drought resistance. To improve the teak germplasm in India, greater research into the assessment of more specific features linked to growth, wood quality, and water-use efficiency in teak in multi-locational provenance experiments is essential.

Data availability
All data generated or analyzed during this study are included in this published article.