Agro-biochemical Traits of Sugarcane Varieties Grown in the Brazilian Semi-arid Region

The objective of this research was to evaluate the productive response associate to biochemical indicators and oxidative enzymes activities involved in the water deficit resistance in eight sugarcane varieties (RB951541, RB931011, RB962962, RB867515, RB763710, RB72454, RB863129, and RB92579) grown in the semi-arid regions of Brazil. Compared to all other varieties, RB763710 was superior in the number of stems m, mean height, and stem diameter, production of whole plant fresh biomass and stem. When biochemical responses were obtained, all sugarcane varieties had a statistically similar solubility and maturity, regardless of the specific maturity rate of each cultivar. The increase in soluble carbohydrate levels occurred in the most stress-sensitive varieties and the variety RB763710 had the highest proline content. A lower general correlation was observed between the production of fresh biomass of stalks and the enzymatic activity. Among the varieties, RB763710 had the highest enzymatic activities which possibly provided greater tolerance to water stress due to the ability to maintain the redox state in the leaves of plants under water deficit. The study of the adaptation mechanisms of sugarcane against water deficit has contributed to the development and diffusion of genotypes tolerant to rainfed conditions, thus contributing to increased productivity even under adverse conditions, allowing maintenance and optimization of the production chains of sugarcane in rainfed regions.


Introduction
The economic, social and energy valorization of sugarcane (Saccharum officinarum L.) has promoted its cropping even in unfavorable and semi-arid regions due to its diverse uses such as energy matrix, sugar-alcohol, animal production and civil construction. Semi-arid regions have several conditions that can limit crop productivity such as water deficit, high temperatures, soil salinity and high luminosity (Choudhary et al., 2018;Pedroso et al., 2018).
Water stress is one of the main factors that reduces crop productivity (Ferreira et al., 2017;González-Chavira et al., 2018). Other types of stress are triggered as a consequence of water stress such as oxidative stress (due to reduced photosynthesis and increased respiration). Oxidative stress can increase the production of reactive oxygen species, such as singlet oxygen, hydrogen peroxide, superoxide and radical hydroxyl groups, which are capable of damaging vital cell structures and organelles, culminating in cell death, depending on the severity of the stress (Iqba et al., 2018).
As a defense mechanism against unfavorable conditions, plants produce antioxidant enzymes and other non-enzymatic compounds that are able to neutralize the damage caused due to stress (Jiang et al., 2016;Marcos et al., 2018). Antioxidative enzymes include catalase, superoxide dismutase, ascorbate peroxidase, polyphenoloxidase, peroxidase and glutathione reductase (Moura et al., 2018).

Method
The experi from febru W; altitude is of the ty respectivel lower to re 24.34 mm demand re worth noti the plants  (Marcos ment programs, ., 2018  According to the results of the soil analysis, the experimental site was fertilized with 181, 142, and 134 kg ha −1 of urea, triple superphosphate, and potassium chloride, respectively based on the Manual of Recommendations for Fertilization for the State of Pernambuco (IPA, 1998). The planting of sugarcane was carried out by the "foot with tip method, using stems sectioned in smaller tops containing three to four buds, aiming at greater uniformity of the plants.
The evaluated varieties were RB951541, RB931011, RB962962, RB867515, RB763710, RB72454, RB863129, and RB92579 chosen for having great representativity in the semi-arid region of Pernambuco (RIDESA, 2010) ( Table 2). The sugarcane seeds were obtained from the Agronomic Institute of Pernambuco, Carpina Sugarcane Experimental Station in agreement with the Interuniversity Network for the Development of the Sugarcane Sector. Sugarcane planting, measurements, and collection of plant material for production, soluble solids content, and biochemical characteristics were carried out in October 2015. At the study site, the number of plants, stems m −1 , average stem height [(n = 10) measured at soil level to the sheath of the first leaf (+1)] and the stem diameter was measured as per Hermann and Câmara (1999). The production of fresh biomass of the stem and entire plant was obtained by weighing the plants present at the study site and expressed as mg ha -1 .
The content of soluble solids (Brix) was evaluated from the sugarcane juice extracted from the internodes of the second and third stem base, end of the stem, penultimate and antepenultimate internodes, and from the entire stem, enabling the monitoring of the maturity index of sugarcane. The broth was extracted with the aid of a mill. Following grinding, the broth was filtered (0.053 mm sieve) to remove bagasse and impurities. A bench refractometer (ABBE-ART-100) was used to determine the Brix using two drops on the refractometer prism.
The leaf samples were collected from the leaves without ribs, frozen in liquid nitrogen and kept at -80 °C for future biochemical analyses of soluble carbohydrates (CHO sol ), proline (PL) and enzymatic activity. The extraction and determination of CHO sol was performed according to Dubois et al. (1956) with modifications: 0.05 jas.ccsenet.org Journal of Agricultural Science Vol. 11, No. 11; g of the frozen material was macerated in 1.3 mL of distilled water and the extract centrifuged at 12,000 × g for 21 min at 4 °C.
Subsequently, 25 μL of the supernatant was diluted in a solution containing 475.0 μL of distilled water, 500.0 μL of 5% phenol, and 2.5 mL of concentrated sulfuric acid. After standing for 10 min, the solution was stirred for a few seconds and then allowed to stand again for 20 min in beakers containing water at room temperature (25 °C). The readings were recorded using a spectrophotometer at 490 nm. The CHO sol content was calculated from a standard curve prepared with 180 μg mL -1 glucose and the results were expressed as mg g -1 fresh leaf mass (FLM).
The extraction and determination of proline (PL) was performed according to a modified method described by Bates (1973). Fresh leaf tissues (0.5 g) were weighed from the central region of the leaf blade, without the ribs and packed in 10 mL of distilled water, followed by placing it in a bath for 1.0 h at 100 °C. The extracts were then filtered twice using Whatman® qualitative filter paper. A 1 mL aliquot of the filtrate, 1 mL of acidic ninhydrin (1.25 g L -1 ninhydrin, 30 ml L -1 of glacial acetic acid, and 20 ml L -1 of 6 M phosphoric acid) were added in a container with lids along with 1 mL of glacial acetic acid. The solution was homogenized and placed in the water bath for 1 h at 100 °C and then in an ice bath for 15 min to interrupt the reaction.
After reaching room temperature, 2 mL of toluene was added and the solution was homogenized for 15 seconds. After 30 min of homogenization, the spectrophotometer readings were recorded at 520 nm. For quantification of the free proline, a standard curve was prepared using proline. The results were expressed in μmol g -1 FLM.
For the enzymatic activities the foliar extract was obtained according to methodology proposed by Silva (1981) and Simões et al. (2015). Estimates of the enzymatic activity of peroxidase were performed using the method described by Urbanek, Kuzniak-Gebarowska, and Herka (1991), using guaiacol and H 2 O 2 as substrates. The activity of the polyphenoloxidase was verified by the oxidation of pyrogallol according to Kar and Mishra (1976) and the catalase activity measured according to the recommendations of Havir and Mchale (1987). Superoxide dismutase activity was measured by using the method proposed by Giannopolitis and Ries (1977), and Beauchamp and Fridovich (1971). The enzymatic activities were expressed in U.A. min -1 g -1 FLM.
The data obtained from productivity, biochemistry, and enzymatic activity were subjected to tests of normality, homoscedasticity, and analysis of variance. The averages were compared by the Scott-Knott test (p ≤ 0.05), using SISVAR software 5.6. In addition, Pearson's correlation between general production data, and proline and enzymatic activities was performed. Log-transformed data was used to construct the scatter plot by main coordinate analysis obtained on the basis of the similarity matrix of the Jaacard Coefficient using PAST 1.9 software.

Results
The sugarcane variety RB763710 was significantly superior in all agronomic variables such as the number of stems m −1 , mean height, and main stem diameter, production of whole plant fresh biomass and stem, followed by RB962962, RB867515, and RB72454 (Table 3).   lyphenoloxidas do not differ by Vol. 11,No. 11; d the highest y elation between independent o vels occurred in n ( high productivity and rapid growth however the variety RB763710 showed the highest average plant height even under water stress conditions, which has a semi-decumbent growth habit. The results observed in this study corroborate with those found by Silva et al. (2011), andOliveira et al. (2015) that showed stem diameters of 2.450 and 2.486 cm for the varieties RB 92579 and RB72454, respectively. In both studies, the varieties were subjected to water stress, similar to this current study.
It should be emphasized that the maturity indices found in this current study represent sugarcane maturity according to the classification of Cesnik and Miocque (2004). Oliveira et al. (2011), andCardozo andSentelhas (2013) showed the negative influence of increased water availability on these varieties; hence, sugarcane plants submitted to rainfed conditions tend to accelerate their maturation. Sales et al. (2012) showed that the CHO sol contents remained constant for the tested varieties and their performance in the deterioration of the starch reserves of the plant when water stressed. Patade et al. (2011) found results different from those observed in this study (Table 4), where there was an increase in the levels of CHO sol in the varieties more sensitive to stress (water and salinity).
Proline has been shown to have a strong correlation with increased drought tolerance (Balestro et al., 2017;Castañeda et al., 2018). However, Mansour and Ali (2017), and Marcos et al. (2018) argued that since proline is produced under stressful conditions, its absence or low production by the plant may indicate less stress.
The accumulation of proline in plant tissues is associated with a reduction in the concentration of toxic ions and the increase of water volume in the cytosol, besides protecting the cell membranes from oxidative stress (Merwad et al., 2018). Proline has been shown to decrease the osmotic potential and, consequently, maintain water and cell turgescence potential near adequate levels, being extremely important for the growth and development of plant tissues (Jungklang et al., 2017;De la Torre-González et al., 2018).
The low correlation between fresh stem biomass production and enzymatic activity in the present study may have been due to the greater tolerance to water stress as a consequence of the ability to maintain the redox state in the leaves of plants under water deficit.
Peroxidase integrates the oxidoreductase group, which catalyzes a large number of oxidative reactions, using peroxide as a substrate, or in some cases oxygen as a hydrogen acceptor; a process of high importance for the adaptation of plants to water stress (Freitas et al., 2008) as also observed in this current study with sugarcane.
Polyphenoloxidase is an enzyme that catalyzes the oxidation reaction of phenols in quinones, acting in an aerobic environment, located in the plastids (Kuwabara & Katoh, 1999). The action of polyphenoloxidase reduces the amount of superoxide, causing oxidative damage in the plants, thus being an indicative of varieties tolerant to water restriction. However, the responses may vary between cultivars, species, tissues analyzed, duration and the magnitude of stress (Campos et al., 2004).
Superoxide dismutase plays an important role in the adaptation and survival of stressed plants, and the first line of defense against reactive oxygen species forming superoxide for hydrogen peroxide, generating lower levels of lipid peroxidation (Scandalios, 2005;Amaro, 2018). Superoxide produced in excess as a result of the stress caused by the water restriction, is extremely reactive and can be transformed into a hydroxyl radical, which is the most harmful of all the radicals, with the capacity to lead to deleterious changes in primary and secondary metabolism and mutations, leading to cell death and in high severity, plant death (Vranova et al., 2002).
The catalase enzyme has amain auxiliary function in the ascorbato-glutathione cycle, acting toward the detoxification caused by the hydrogen peroxide, in the cells of the plants (Sheikh-Mohamadi et al., 2018). Catalase activity increases according to the amount of hydrogen peroxide within the cells . The values observed in this current study were low because sugarcane is a C4 plant possessing minimum photorespiratory ability; contrastingly C3 plants are more involved in the removal of hydrogen peroxide (Tolbert et al., 1969). Benesová et al. (2012) observed that the variety more tolerant to water stress had greater catalase activity and vice versa thus corroborating with the results observed in this current study.