Effects of Life-form and Plants Functional Traits on Carbon, Nitrogen and Sulfur Distributed in Different Parts in Dry Region of Western India

Background: Plants adapt to adverse environmental conditions accumulate varying concentrations of carbon (C), nitrogen (N) and sulfur (S) compounds to cope up with adverse climatic conditions. Carbon, N and S concentrations were determined in roots, stem and leaves of 33 species of trees/shrubs with objectives to observe the effects of life-form and plants functional traits, and select species with high concentration of these elements for their utilization in afforestation and medicinal uses. Results: Concentrations of C, N, and S and C: N and N: S ratio varied (P<0.05) between species, organs, life-forms and functional traits (legume vs non-legume). These variables were higher (except C in roots and stem) in trees than shrubs, and in leguminous than non-leguminous species. Non-leguminous species showed high S content and low N: S ratio. Antagonistic and synergistic relations were observed between C and N, and N and S concentration respectively. Species showed varying potential in assimilating carbon by regulating uptake and accumulation of these elements in different organs making them adapt to the habitats affected by drought and salinity. We observed strong plant size/life-form effects on C and N content and C: N and N: S ratios and of function on S content. Conclusions: Life-form/size and varying functions of the species determined C: nutrient ratio and elemental composition and helped adapting varying environmental stresses. This study assist in selecting species of high carbon, nitrogen and S content to utilize them in afforesting the areas affected by water and salt stresses, increased carbon storage and species with high S/N content in medicinal uses. C. H. antidyncenterica, mysorensis and Z. nummularia observed high in average C content, whereas Capparis decidua, S. oleoides, Dypterigium glaucum and Euphorbia caducifolia observed high in S content. This study offered an opportunity for the selection of species with high carbon, nitrogen and S content for their use in greening the areas affected by water and salt stresses, increased carbon storage and in pharmaceuticals particularly the species with high S-N content.


Background
The pathways of sulfur (S), carbon (C) and nitrogen (N) are well coordinated to maintain the physiological functions in plants ( Giordano et al. 2000; Kopriva et al. 2002). Capacity of the plants to uptake and reallocate the element and consequently changes in the elemental composition and stoichiometry of the plant system are in uenced by environmental conditions, altitude, soil texture and mineral elements availability (He et al. 2016;Shedayi et al. 2016). These mineral elements are reallocated by accumulation of metabolites along different pathways including O-acetylserine to serine to glycine, and are further channeled together with the nitrogen-rich compound glutamine into allantoin (Nikiforova et al. 2005). Likewise synthesis of cysteine from sul de and O-acetyl-L-serine (OAS) is a reaction interconnecting sulfate, nitrogen, and carbon assimilation (Kopriva et al. 2002). De ciency of any such element disrupts C: N: S ratio altering metabolic processes like decrease in cellular carbohydrate levels and respiration rate and breakdown of many complex organic molecules (Nikoforova et al. 2005; Dubousset et al. 2009) affecting biomass allocation (Lin and Wu 2004).
Allocation of these elements in above-and below-ground components of the plants is one of the key processes of their cycling and depends on species and soil or habitat types (Chen et al. 2015). Carbon allocation to shoots and roots is mediated by nitrogen supply via regulating cytokinins and sucrose production (Van der Werf and Nagel 1996). Wood C content also vary substantially among species ranging from 41.9-51.6%, but does not relate to ecological (i.e. wood density, maximum tree height), demographic (i.e. relative growth rate, mortality rate) or phylogentic traits (Martin and Thomas 2011).
Some trees and shrubs show high ability to x atmospheric carbon di oxide into their biomass with C content ranging from 51.66% in Eugenia caryophyllata to 47.77% in Rosamarinus o cinalis (Maiti et al. 2015). Carbon concentration also vary with tree characteristics and tree organs like living branch > bark > foliage > dead branch > stem in aboveground and large roots > stumps > thick roots > medium roots > small roots in belowground organs ). While C and N are used for structural macromolecules, sulfur has critical roles in the catalytic or electrochemical functions of the biomolecules in cells. Increased carbon concentration and total biomass is favoured by increased rate of carbon sequestration (i.e. rate of photosynthesis), whereas there is reduction in growth and biomass productivity under drought and a typical de ciency or insu cient supply of S or N. Sulfur de ciency also induces imbalance between carbon and nitrogen indicating importance of this nutrient on growth and productivity (Schonhof et al. 2007) and even tolerances to biotic and abiotic stresses in plants ( Khan et al. 2014).
Species occurring in dry areas have ecological advantages of growing under varying biotic (human and livestock) and abiotic (limited soil water and increased salts) stresses and maintain higher abundances than the species that do not possess the characteristics of drought or salinity/alkalinity tolerances. In the context of the role of plants in capturing carbon dioxide from atmosphere and accumulate nitrogen and S metabolites in stressful conditions of dry areas, present study focused on (i) to determine C, N and S concentrations in various native and exotic species growing in Rajasthan, India; and (ii) to screen tree and shrub species with high carbon, sulfur and nitrogen content for increased carbon sequestration and adaptability in water/salt stressed region for their use in future afforestation programmes.

Site conditions
The study area is Rajasthan, which lies between latitudes 23º 3' and 30º 12' N and longitudes 69º 30' and 78º 17' E. Because of its location in the western part of India along with varying topography, Rajasthan exhibits varying climate. The rocky Aravali, the western arid plains, the eastern fertile plains etc., experience different climatic conditions. Long term average value of annual rainfall in Rajasthan is 575.1 mm. However, it varies widely among the districts ranging from 185.5 mm in Jaisalmer to 950.3 mm in Banswara (Fig. 1). Average annual rainfall for last 10 years is 663.3 mm with variations from 393.5 mm in 2009 to 851.8 mm in 2011 (Fig. 1). Average rainfall decreases from East to West and from South-west to North-east. There is marked decrease in rainfall west of the Aravalli range making the western Rajasthan arid.

Plant sampling and analysis
Plant samples were collected from exiting trees and shrubs species during 2010-2013. A total of 180 samples were taken from 19 trees and 14 shrubs species. After felling of the trees of different species, leaf, stem and root samples were collected. These samples were brought to laboratory and washed under the tap water for any soil and contaminants adhered to the samples and dried in hot air oven at 60-80°C for a constant weight. These dried plant samples were ground for a homogenous powder using a Tonko make Wiley Mill (no. 40 mesh). Powdered samples were then analyzed for Carbon (C), Nitrogen (N) and Sulfur (S) using CNS analyzer model (Flemetar make, Model -Vario EL Cube). In this about 20 mg ne ground wood/plant leaf samples were weighed using Mettler Toledo micro balance and the sample was put to the sampler of the CNS analyzer for combustion and C, N and S analysis using Sulphanilamide as the standard.

Statistical analysis
The data collected were statistically analyzed using SPSS statistical package version 8.0 for window 2000. Carbon, nitrogen and sulfur content in different parts like root, stem and leaves in different species were analyzed by two-way ANOVA, where tree/shrub species and their different organs were the main factors and number of trees/shrubs were the replicates. Data were also analyzed considering tree and shrubs as plant habits as well as considering leguminous and non-leguminous species. Data were also subjected to Pearson's correlation and regression analysis to nd relationships between different mineral constituents. The least signi cant difference test was used to compare treatments at the P < 0.05 levels.

Sulfur concentration
Concentration of S ranged between 0.01% and 1.17% in roots, 0.01% and 2.07% in stem, and 0.04% and 2.28% in leaves across the species and plant habits. Among the organs, average S concentration varied signi cantly (P < 0.05) and it was highest in leaves (0.30 ± 0.09%) followed by roots (0.26 ± 0.02%) and stem (0.21 ± 0.02%). Almost 19 species under study showed highest concentration of S in leaves, whereas 10 species namely A. catechu, A. senegal, P. cineraria, P. juli ora, A. pseudotomentosa, C. polygonoides, C. procera, D. cineria, D. glaucum, H. salicornicum had highest S concentration in roots. Four species like E. caducifolia, H. antidyncenterica, L. pyrotechnica and R. mysorensis showed greater S in stem than in the other organs. Though not signi cant (P > 0.05), S concentration was relatively greater in all three organs of trees as compared to those in the shrub species (Fig. 4a). However, S concentration was signi cantly (P < 0.05) greater in all these organs of non-leguminous species as compared to leguminous species when functional aspect was considered (Fig. 4b). Among the species Capparis decidua, S. oleoides, Dypterigium glaucum and Euphorbia caducifolia showed signi cantly (P < 0.05) higher average S concentration than the other species. Out of 33 species studied, 9 species (4 tree species and 5 shrub species) indicated above average (0.24%) sulfur concentration.
Carbon: N and N: S ratios Carbon to nitrogen ratio ranged from 18.24 for Adhatoda vasica to 79.27 for P. juli ora with an average value of 32.42. Nitrogen to sulfur ratio varied from 0.74 for Salvadora oleoides to 36.68 in case of C. mopane with an average value of 12.76 across the species (Fig. 5). Both C: N and N: S ratios were greater for tree species than for shrub species between plant habits. These ratios were greater in leguminous species than the non-leguminous species. Seven species of shrubs and 11 species of trees showed below average C: N ratio (Fig. 5). Almost 8 tree species and 5 shrub species showed C: N ratio below 35. P. juli ora showed highest (P < 0.05) C: N ratio. Anogeissus pendula, B. racemosa, C mopane, H. salicornicum, H. antidycenterica, and Z. nummularia showed highest (P < 0.05) average N: S ratio (> 20). S. oleoides showed lowest N: S ratio, but it did not differed in this ratio for other 26 species. Species with N:S ratio below 10 were A. tortilis, C. decidua, Maytenus emarginata, P. cineraria, S. oleoides, Adhatoda vasica, B. aegyptiaca, C. polygonoides, C. procera, D. glaucum, E. caducufolia, L. pyrotechnica, which come naturally in the area in uenced by drought or salinity.

Statistical relations
Average stem carbon in plant was negatively correlated to root (r=-0.160, P < 0.05), stem (r=-0.199, P < 0.01) and leaf (r=-0.282, P < 0.01) nitrogen and leaf (r=-0.193, P < 0.05) sulfur and positively correlated to C: N ratio (r = 0.331, P < 0.01). Likewise leaf C concentration showed negative relationships with root Among different organs, carbon, N and S concentration were observed in the descending order of Stem > leaves > root, leaves > roots > Stem and leaves > root > stem respectively, across the species. Global analysis of plant carbon content also indicated highest C content in stem, whereas leaves showed greater C than roots (Ma et al. 2017). Major contribution on interspecies variation in these elements among the species was due to differences in the chemical constituents of wood, i.e. cellulose, lignin and non-

Conclusion And Recommendations
Present study illustrates large variability in C, N and S concentration between trees/shrubs species and their different organs (roots, stem and leaves) as well. As adaptation characteristics towards drought and salinity stresses, these species regulated concentration and stoichiometry of these elements in the region.
Increased C: N and N: S ratios were found related to increase in plant size (tree vs shrubs). However, increased concentration of S and reduced N: S ratio in non-leguminous plants showed their adaptability of growing in habitats affected by drought and salinity stresses. Therefore, different tree and shrubs species have different potential to assimilate carbon by regulating uptake and accumulation of sulfur, nitrogen, and carbon in different organs. A. leucopheloea, A. nilotica, B. monosperma, C. mopane, H. binata, P. cineraria, P. juli ora, A. jaquemontii, B. aegyptiaca, C. polygonoides, E. caducifolia, H. antidyncenterica, R. mysorensis and Z. nummularia observed high in average C content, whereas Capparis decidua, S. oleoides, Dypterigium glaucum and Euphorbia caducifolia observed high in S content. This study offered an opportunity for the selection of species with high carbon, nitrogen and S content for their use in greening the areas affected by water and salt stresses, increased carbon storage and in pharmaceuticals particularly the species with high S-N content.

Availability of data
The datasets used and/or analyzed during the current study are available from the corresponding author on request.    Variations in sulfur concentration in different organs of the plants due to plant habit (tree vs shrubs, a) and functional traits (leguminous vs non-leguminous, b) in Rajasthan, India Figure 6 Relationship between average concentrations of carbon and nitrogen (a) and nitrogen and sulfur (b) in plant system across the species and plant organs