Response of sugar cane to limitation hydric and nitrogen dose

The great social and economic importance of sugarcane makes it critical to understand the responses of the crop to adverse stress. This study was carried out to characterize the morpho-physiological index of sugarcane cultivated under different levels of water replacement with four replicates, corresponding to five levels of water replacement (100, 75, 50, 25 and 0% of soil humidity at field capacity) and either associated or not with a nitrogen source (100 kg ha -1 de N). Leaf area, leaf water potential, gas exchange, and chlorophyll fluorescence were determined at different stages of plant development. Suboptimal maintenance of turgor pressure by water potential reduced the photosynthetically active area of sugarcane submitted to hydric deficit. A water replacement of 75% in association with nitrogen promoted optimal maintenance of the photosynthetic process.


INTRODUCTION
Sugarcane is a major crop grown in Brazil due to its great socioeconomic importance.According to the FAO (2013), the cultivated area with sugarcane was greater than 9.4 million hectares, from which 670.76 million tons were harvested in 2012.In Brazil, the Midwest Central region is responsible for around 60% of national production (UNICA, 2013).The amount produced by area, however, varies with water availability.Hydric stress is the major limiting abiotic factor in world agricultural production.Responses to production are dependent on the development stage at the time of stress (Çakir, 2004) as well as the frequency and intensity of the hydric stress (Cattivelli et al., 2008).In the case of sugarcane, hydric and nutritional deficits affect productivity by reducing photosynthetic capacity; however, the interaction between these stresses is still *Corresponding author.E-mail: marconibt@gmail.com.Tel: +55-64-36205636.Fax: +55-64-36205636.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License not well understood (Gava et al., 2010) and in most cases they can easily be confounded.
The physiological response mechanisms to hydric stress in plants suggest a direct perception of hydric stress by roots that then produce root signals.These promote reductions in the aerial portion, roots, absorption of water and nutrients, and capture of radiation and CO 2 by the leaves.These changes can reduce resource use such as transpiration and radiation (Sadras, 2009).According to Lopes et al. (2011), hydric deficiency induces a low water state in tissues and loss of cell turgidity, besides interfering with nutrient absorption and osmotic stress and reducing transpiration by thermal stress.
Nitrogen deficiency interferes with the activity of phosphoenolpyruvate carboxylase (PEPcase) and ribulose-1, 5-bisphosphate carboxylase/oxygenase (RUBISCO) (Ranjith et al., 1995;Meinzer and Zhu, 1998).Hydric deficit also affects many physiological processes, such as stomatal closing, consequently reducing the supply of CO 2 for photosynthesis, increasing the diffusive resistance to water vapour, reducing transpiration, and affecting thermal energy dissipation and the transport of nutrients by mass flow, among others (Wu and Campbell 2007).Many of these effects represent mechanisms of adaptation of plants to their environment (Heschel and Riginos, 2005).
Thus, irrigation and fertirrigation systems have been used to maximize water usage and minimize the effects of deficiencies in water and nutrients during crop development (Barbosa et al., 2012).Drip irrigation systems are used to apply water with high frequency and low intensity at specific points over a cultivated area, allowing maintenance of a constant soil humidity in the plant root zone (Vekariya et al., 2011).Supplying water and nutrients directly to the root system characterizes subsurface drip irrigation (SDI) for the high use of resources by plants (Oliveira et al., 2014).
Fertirrigation practices allow splitting the manure during crop development, avoiding loss of nutrients by leaching.Souza et al. (2009) revealed that modern fertirrigation practices should consider environmental sustainability and rigorous management of hydric resources.In addition, the nitrogen content of plant is improved by a SDI supply, assigned the major availability of nutrients in soil or capacity of root absorption (Kraiser et al., 2011).
Knowledge about sugarcane responses is essential to crop management, making it important to obtain information about the efficiency of water use by plants and the viability of sugarcane cultivation (Azevedo et al., 2011).In this way, the objective of this study was to understand the physiological characteristics and development of leaf in response to water stress and nitrogen in sugarcane cultivated under different levels of water replacement (irrigation) by subsurface drip with or without a nitrogen supply.

MATERIALS AND METHODS
This study was conducted to complement the survey conducted by Oliveira et al. (2014) in order to achieve understanding of the physiological behavior of sugarcane plants subjected to different levels of water replacement (irrigation) subsurface drip with and without nitrogen supply.Sugarcane, RB 85-5453 variety, was cultivated from March 2011 until April 2012 in an experimental area at Institute Federal Goiano -Campus Rio Verde, Goias state, 720 m in altitude and soft-wavy relief (6% declivity).The climate in the region is classified by Köppen and Geiger (1928) as Aw (tropical), with the rainy season occurring from October to May, and June to September corresponding to the dry season.The average annual temperature ranges from 20 to 35°C and annual rainfall varies from 1500 to 1800 mm.
The experiment was carried out in randomized complete blocks in a 5 x 2 factorial scheme, with four replicates.The treatments consisted of five levels of water replacement (100, 75, 50, 25 and 0% of soil humidity at field capacity) and two conditions of fertilizer supply (0 and 100 kg ha -1 of N) in the form of urea.The experimental plots were set up in a double row composed of three grooves (W-shaped) 8 m long, with a spacing of 1.8 m by 0.4 m between the lines, totalling 35.2 m 2 per plot.
A subsurface drip irrigation system was used for the water replacement treatments.The drip tube, (Dripnet 16150 model) with a thin wall, 1 bar service pressure and 1.0 L h -1 nominal flow, was buried at a depth of 0.20 m below the soil surface in the middle of the double row with a spacing of 0.50 m between drippers.
The water volume applied in the 100% water replacement treatment was based on increasing soil humidity until field capacity was reached.With the other treatments, a water blade was applied considering the water replacement of the 0, 25, 50 and 75%.At the end of the experiment, the total volume of water was 0, 126, 252, 378 and 504 mm accounting for 0, 25, 50, 75 and 100% water replacement, respectively.A nitrogen supply was added to the irrigation water (fertirrigation) and divided into ten applications during crop development, while one portion of potassium manure was added to planting grooves, representing 30% of the total, and the remainder was applied via the irrigation water.In the treatment with 0% water replacement, fertilizers were comprised by broadcast application.
The irrigation was carried out with a tensiometry puncture digital base at 0.1 kPa of sensibility, being a tensiometric metal rod installed at depths of 0.20, 0.40, 0.60 and 0.80 m and a distance of 0.15, 0.30, 0.45 and 0.60 m from the drip tube.The soil matrix potential (Ψm) was collected daily.A critical tension of 50 kPa was used to determine the need for irrigation.The physical-hydric characteristics of the soil were determined from a water retention  Doorenbos and Kassam, 1979): budding and establishment (Kc = 0.6); establishment and tillering (Kc = from 0.9 to 1.1); maximum growth (Kc = 1.3); maturation (Kc = from 0.7 a 0.9).Source: INMET -Rio Verde, GO, Brazil.
curve.An equation was developed according to Van Genuchten (1980) to convert the Ψm to water content in the soil (θ), minimizing the square sum of the deviation using SWRC software (Dourado-Neto et al., 2000), and so obtaining the empirical parameters of adjustment used in the equation 1: (1) Where: θwater content of soil, g g -3 ; Ψmmatrix potential, mca.
The pluviometric rainfall was used to estimate the decendial water balance, calculating the hydric deficit (HD) during the experiment (March 2011 until April 2012) using the method of Thornthwaite and Mather (1957), in which the Reference Evapotranspiration (ET0) was calculated according to the Penman-Monteith (Monteith, 1973) equation.These results are shown in Figure 1.Total evapotranspiration during crop development was 1,549 mm and rainfall was 1,618 mm In the useful area (one square meter) around the main line three plants were randomly chosen to determine gas exchange, chlorophyll fluorescence, leaf water potential (Ψw), leaf area and number of leaves.
The gas exchange rates of the plants were evaluated at 300 and 410 days after planting (DAP) to obtain the photosynthetic rate (A, μmol m -2 s -1 ), transpiration rate (E, mmol m -2 s -1 ), stomatal conductance (gs, mol H2O m -2 s -1 ) and ratio of internal to external CO2 concentration (Ci/Ca).The water use efficiency (WUE, mmol CO2 mol H2O -1 ) was calculated from the A/E ratio.Measurements were performed on completely expanded leafs using a portable photosynthesis system with a LCi Light Systems (ADC Bioscientific, Herts, England) using a light source emitting a photon flux density of 1000 μmol m -2 s -1 .

Statistical analysis
Result were evaluated with variance using an F-test at a probability of 0.05.Where significance was detected, a linear and quadratic polynomial regression to water replacement and nitrogen supply were compared by Tukey test at a probability of 0.05.

RESULTS AND DISCUSSION
Analysis of variance showed that water replacement (WR) significantly altered all variables analysed except transpiration rate, stomatal conductance and water use efficiency (WUE) at 410, and 300 days after planting(DAP), respectively (Table 1), whereas the nitrogen (N) supply promoted significant differences in photosynthetic rate (A), stomatal conductance (g s ) and  2A).However, studies on two genotypes of sugarcane showed accelerated reductions in CO 2 assimilation and transpiration of plants submitted to hydric deficit during sucrose accumulation as a response to decreasing stomatal conductance (Machado et al., 2009).
Photosynthetic rate (A) decreased by 8.53 μmol m -2 s -1 with 54,12% of water replacement, followed by increases of 2.83 μmol m -2 s -1 and 13,68 μmol m -2 s -1 for 75 and 100% WR, respectively, when we consider the water replacement inside each nitrogen doses at 410 DAP.However in the absence of nitrogen no differences among levels of water replacement were verified (Figure 2B).
When we considered the nitrogen dose inside each level of water replacement for A (Table 2), there were no statistical differences among the doses at 25% water replacement.At WR levels of 0 and 100%, plants that received nitrogen fertilizer showed a major A. However, at 50 and 75% WR levels the plants without nitrogen supply showed greater photosynthetic rate.The transpiration rate (E) evaluated at 300 DAP showed a quadratic response to water replacement levels.The maximum E of 5.72 mmol m -2 s -1 was obtained.At 44% of WR, from which reductions of 0.29, 7.74 and 25.56% in response to increases in water replacement to 50, 75 and 100%, respectively (Figure 2C).The E at 410 DAP increased by 25.09% with N application (Table 3).Increases in leaf transpiration favour absorption of solute from soil leading to improved nutrient assimilation, in this case supplied in abundance via the irrigation system.Biomass production is strongly linked to transpiration; maximizing absorption of humidity from the soil is the main point to improve crop yield under conditions of water stress conditions (Blum, 2009).
Plants fertilized with N showed high photosynthetic rates (A) and WUE at 300 DAP (Table 3).The nitrogen supply induced an increase of 14.75% WUE when compared to plants that did not receive N (Table 3).
Considering water replacement inside each nitrogen level was verified responses of WUE at 410 DAP, as N application, adjusting to quadratic equation (Figure 2D)., and an increase of 2.47 fold at 100% water replacement.Studies verifying the relationship of sugarcane cultivated in different environments and at different stages of development to water found 4.4 µmol of CO 2 fixed to each water molecule released by transpiration (Endres et al., 2010), as observed in the present study (Figure 2D).
On the other hand, splitting nitrogen doses in each water replacement level at 410 DAP produced significant differences only at 75% water replacement, as a decrease of 11.43% in WUE with nutrient supply (Table 2).The efficiency of water use to crop yield has been used to indicate to increasing plant productivity per unit of water when compared to dry cultivation (Blum, 2009).In this way, is possible to demonstrate that water supply during crop development maintains sufficient humidity for the photosynthetic process.However, plants growing with 0% water replacement presented high resistance to drought, reaching 14.35 mmol CO 2 mol H 2 O -1 of WUE at 410 DAP (Figure 2D) Maintaining the photosynthetic process is functionally linked to stomatal conductance (g s ), directly linked to the transpiration and photosynthetic rates of sugarcane plants.Based on the quadratic behaviour of g s as a response to water replacement at 300 DAP, reaching a maximum response with 48.33% water replacement at a  g s of 0.35 mmol m -2 s -1 followed by a 22.75% decrease compared to 100% water replacement (Figure 2E).These results reinforce the idea of the stomata being associated with morphological characteristics as tolerance mechanisms against hydric deficit (Endres et al., 2010).When N was applied, a 27.8% reduction in g s was verified at 300 DAP (Table 3).
Neither regression model linear or quadratic was adjusted to the ratio of internal to external CO 2 concentration (Ci/Ca) in sugarcane leaves at 300 DAP, showing analogous efficiencies on stomatic absorption.However, the results observed at different water replacement levels showed that Ci/Ca in the stomatal chamber at 410 DAP presented a quadratic behaviour (Figure 2 F).Ci/Ca reached its maximum value, 0.793, with 56.0% water replacement and decreased by 2.28 and 12.20% when water replacement was raised to 75 and 100%, respectively.
Significant decreases in internal concentration (Ci) should result in reduced photosynthetic rates in response to reductions in CO 2 concentration needed for the activity of the enzymatic complex of PEPC and RUBISCO (Parry et al., 2011).Whereas low values of Ci can stimulate stomatal opening, allowing a major influx of CO 2 into the substomatal cavity (Raschke, 1979) and permitting an equilibrium between consumption and entry of CO 2 , keeping Ci constant.In this way, adequate levels of water replacement could promote excitation of stomata in order to maintain CO 2 at a high concentration in the stomatic chamber.
A significant effect of the nitrogen supply was observed on Ci/Ca at 410 DAP, representing an increase of 13.75% compared to no nitrogen treatment (Table 3).
Reductions in transpiration and the photosynthetic rate of sugarcane at high levels of water replacement when compared to other treatments can be associated with rainfall during the experimental period (Figure 1).
According to Ghannoum (2009), increasing the CO 2 concentration after hydric stress can partially recover photosynthesis limits under pre-stress conditions in plants with C4 metabolism.So, the related g s and E responses to plants were not impeded by water replacement at 410 DAP (Table 1), verifying the high capacity for restoration of photosynthetic machinery and consequently the photosynthetic activity of plants submitted to hydric deficit (Figure 2B).Water replacement significantly interfered with NPQ at 380 and 410 DAP.The statistical difference in NPQ was verified at 380 DAP as a response to nitrogen supply (Table 3).Evaluating the WR x N interaction, significant differences in ETR were observed at 380 and 410 DAP (Table 4).However, water and nitrogen supply did not interfere with the potential quantum yield (Fv/Fm) or effective quantum yield (ΔF/Fm') of sugarcane in either evaluation (Table 4).This shows the plant's ability to maintain similar values of Fv/Fm under hydric stress, indicating the efficiency of energy use in the photochemical process (Silva et al., 2007) and corroborating Gonçalves et al. (2009), where sugarcane growing under moderate or severe hydric deficit produced little reduction in potential photochemical efficiency.
Productivity responses of sugarcane to water replacement in association with nitrogen manure are well known (Wiedenfeld and Enciso, 2008).Considering the physiological responses to combination management, it is important to emphasize the efficiency of the photosynthetic machinery with regard to nitrogen use when directly available in the soil solution.
The electron transport rate (ETR) of sugarcane showed a quadratic behaviour when splitting WR in each nitrogen dose at 380 DAP (Figure 3A).According to the regression equation, when water replacement associated with nitrogen supply was at 51.47% of maximum, values of ETR were 22.58% higher than with dry treatment (0% water replacement).However, in treatments without a nitrogen supply the ETR response was more efficient but required 85.08% water replacement to reach a maximum value.Usually, plants submitted to hydric deficit shown inadequate dissipation of electrons produced by the electron transport chain, due a small carbon assimilation rate conducive to overproduction of reactive oxygen species (ROS) (Edreva, 2005).Table 2 verifies the negative interference of nitrogen application on the ETR of sugarcane at 380 DAP with 75 and 100% water replacement levels, with reductions of 32.35 and 29.11%, respectively.The ETR of sugarcane at 410 DAP adjusted to a quadratic equation when splitting water replacement levels in each nitrogen dose.The nitrogen supply showed increasing development at 46.22% water replacement, followed by a decrease with further increases in water replacement.Treatment without a supply of N did not present significant differences among water replacement levels (Figure 3B).Splitting the nitrogen in each water replacement level, the ETR of sugarcane was significantly interfered with at 410 DAP only at 25 and 50% water replacement, triggering increases of 19.33 and 67.33%, respectively, when nitrogen was supplied (Table 2).
Non-photochemical dissipation (NPQ) is dependent on high energy and is related to the proton concentration inside thylakoids, which induce thermal energy dissipation by the xanthophyl cycle (Taiz and Zeiger, 2013).Responses of sugarcane to water replacement showed a linear rise at 380 DAP, triggering an increase of 0.9% in NPQ with a 1% increase in water replacement (Figure 3C).At 410 DAP a variable behaviour was observed in NPQ in response to the water replacement level (Figure 3D).Excess photons should be a main factor in photo inhibition, altering the quantum efficiency of photosystem II (Taiz and Zeiger, 2013).The NPQ data showed a slight increase in response to water replacement, indicating their capacity to activate protection mechanisms against light or photochemical damage.The nitrogen supply promoted a 34.04% reduction in NPQ at 380 DAP in plants supplied with nitrogen (Table 3).
The responses in terms of leaf area (LA) and leaf number (LN) of sugarcane to water replacement showed a linear response.The leaf area increased 13.96 cm 2 for each interval of water replacement (Figure 4A) and rose 3.21% in terms of number of leaves (Figure 4B).Hydric deficiencies caused levels of leaf senescence and restricted the emergence of new leaves.The extent of these alterations is a direct consequence of the degree of hydric stress (Smit and Singels, 2006).
The development of leaf area, that is, photosynthetic active structure, is directly correlated to a high capacity for photoassimilation.Oliveira et al. (2014) observed that with the makeup water and nitrogen there was an increase in the productivity of stems, as increased total biomass and aerial part.According to Inman-Bamber and Smith (2005) and Ghannoum (2009), the morphological characteristics modified by hydric stress are of great importance to increasing plant productivity.
The leaf water potential (Ψ w ) results indicated a linear reduction when water replacement was reduced, reaching its lowest point in plants that did not receive irrigation (-0.18 MPa) (Figure 5).
Water potential provides a relative index of hydric stress (Taiz and Zeiger, 2013).According to Inman-Bamber and Jager (1986), cellular extension is practically insignificant as Ψ w approaches -1.3 MPa.Factors promoting reductions in water potential when plants are submitted to hydric deficit (Figure 5) could be related to a lower pressure of water turgor inside cells due to solute accumulation in the cytoplasm as a way to sustain the turgor pressure, elevating the tolerance of the plant to water deficiency (Inman-Bamber and Smith, 2005).

Conclusion
The low maintenance of turgor pressure in cells as Water replacement (%) evidenced by reductions in leaf water potential promoted severe reductions in the photosynthetically active area of sugarcane when submitted to hydric deficit.Association of the gas exchange rate and chlorophyll a fluorescence of sugarcane plants submitted to water replacement correlated with major development of leaf area, as shown by the high capacity to photoassimilate production.
A water replacement of 75% associated with nitrogen supply via an irrigation system positively influenced maintenance of the photosynthetic process.In addition, it promoted efficient control of leaf transpiration, permitting a high photosynthetic rate that induces efficient use of water.

Figure 2 .
Figure 2. Photosynthetic rate (A) at 300 DAP (A) and 410 DAP (B); transpiration rate (E) at 300 DAP (C); water use efficiency (WUE) at 410 DAP (D); stomatal conductance (gs) at 300 DAP (E); and ratio of internal to external CO2 concentration (Ci/Ca) at 410 DAP (F) of sugarcane submitted to different levels of water replacement, with or without a nitrogen supply.
the same letter in a row, as a function of nitrogen supply, are not significantly different according to Tukey test (0.05).

Figure 3 .
Figure 3. Electron transport rate (ETR) at 380 DAP (A) and 410 DAP (B); non-photochemical dissipation (NPQ) at 380 DAP (C) and 410 DAP (D) of sugarcane submitted to different levels of water replacement, with or without a nitrogen supply.

Figure 4 .Figure 5 .
Figure 4. Leaf area (A) and leaf number (B) of sugarcane submitted to different levels of water replacement.

Table 1 .
Analysis of variance summary of leaf water potential (Ψw), leaf area, number of leaves, photosynthetic rate (A), transpiration rate (E), stomatal conductance (gs), ratio of internal to external CO2 concentration (Ci/Ca) and water use efficiency (WUE) of sugarcane submitted to different levels of water replacement (WR), with or without a nitrogen (N) supply, to 300 and 410 days post-application.
1 Significance: **p < 0.01, * p < 0.05, ns not significant, according to F test.WUE at 300 DAP and the ratio of internal to external CO 2 concentration (C i /C a ) at 410 DAP.For the interaction WR x N, differences were verified at 410 DAP in A and WUE.

Table 2 .
Photosynthetic rate (A), water use efficiency (WUE) and electron transport rate (ETR) in sugarcane plants as a response to different levels of water replacement, with or without a nitrogen supply.Data are mean of evaluation realized at 380 and 410 DAP.

Table 3 .
Photosynthetic rate (A), transpiration rate (E), stomatal conductance (gs), ratio of internal to external CO2 concentration (Ci/Ca), water use efficiency (WUE) and non-photochemical dissipation (NPQ) in sugarcane plants as a response to nitrogen supply.
a 6.10

Table 4 .
Analysis of variance summary of potential quantum yield (Fv/Fm), effective quantum yield (ΔF/Fm'), nonphotochemical dissipation (NPQ) and electron transport rate (ETR) of sugarcane submitted to different levels of water replacement (WR), with or without a nitrogen (N) supply.