Morphophysiology, morphoanatomy, and grain yield under field

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Aug 27, 2013 - Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, ... DKB 390 (tolerant) surpassed hybrid BRS 1030 (sensitive).
Acta Physiol Plant (2013) 35:3201–3211 DOI 10.1007/s11738-013-1355-1

ORIGINAL PAPER

Morphophysiology, morphoanatomy, and grain yield under field conditions for two maize hybrids with contrasting response to drought stress T. Correˆa de Souza • E. Mauro de Castro • P. Ce´sar Magalha˜es • L. De Oliveira Lino • E. Trindade Alves • P. Emı´lio Pereira de Albuquerque

Received: 4 April 2013 / Revised: 19 June 2013 / Accepted: 14 August 2013 / Published online: 27 August 2013 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2013

Abstract In the northern region of the state of Minas Gerais, lack of rainfall limits crop production in the field, which is possible only with irrigation. Agricultural and physiological practices have been intensively searched to overcome drought effects and consequently increase production. In this context, the objective of this study was to characterize morphophysiological and morphoanatomical changes and evaluate the attributes of grain yield under field conditions in two hybrids contrasting for drought tolerance. The experiment was carried out for 2 years (2010 and 2011) and the water deficit was imposed by stopping irrigation for 22 days at the pre-flowering stage. At the end of the stress treatment, leaf and root anatomy and morphophysiological characteristics (leaf water potential, chlorophyll content, percentage of dry leaves, Communicated by P. Sowinski.

leaf area, stomatal conductance, chlorophyll fluorescence, and anthesis-silking interval) were evaluated. For a better interpretation of tolerance of the hybrids in the evaluated characteristics, an index was used stress index. Hybrid DKB 390 (tolerant) surpassed hybrid BRS 1030 (sensitive) in grain yield. Furthermore, it presented lower percentage of dry leaves, higher flowering synchronization, higher stomatal conductance, and higher Fv/Fm relationship. In the root, DKB 390 showed higher amount of aerenchyma in the cortex, an increase of exodermis width, and numerous metaxylem with smaller diameter. In the leaf, it presented higher number of stomata and smaller distance between the vascular bundles in the leaf blade. The study concluded that significant morphophysiological and morphoanatomical changes, which are related to drought tolerance, occurred in DKB 390, leading to a higher yield in the field.

T. C. de Souza (&)  E. M. de Castro  L. De Oliveira Lino  E. Trindade Alves Departamento de Biologia,Setor de Fisiologia Vegetal, Universidade Federal de Lavras, Campus Universita´rio, caixa postal 37, Lavras, MG CEP 37200-000, Brazil e-mail: [email protected]

Keywords Zea mays L.  Water stress  Leaf anatomy  Root anatomy  Stomatal conductance  Harvest index

E. M. de Castro e-mail: [email protected]

Introduction

L. De Oliveira Lino e-mail: [email protected]

A great number of studies have been carried out to evaluate the effects of climate changes on the agriculture. Changes in water availability are one of the major factors of climate changes (Asharaf 2010). Under low soil water availability conditions, a decrease of leaf water content occurs, leading to water deficit. A quick response to this deficit is the closing of stomata, which limits the conductance of gases in the leaves and consequently limits photosynthesis and yield (Mutava et al. 2011).

E. Trindade Alves e-mail: [email protected] P. Ce´sar Magalha˜es  P. E. P. de Albuquerque Centro Nacional de Pesquisa de Milho e Sorgo, caixa postal 151, Sete Lagoas, MG CEP 35701-970, Brazil e-mail: [email protected] P. E. P. de Albuquerque e-mail: [email protected]

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The impact of water stress can be minimized through genetic enhancement geared toward achieving drought tolerance. Despite the sensitivity of maize to drought, promising results have been found in the search for tolerant genotypes (Makumbi et al. 2011; Monneveux et al. 2006). This genetic variability in maize has been found through the understanding of genetic and physiological responses toward stress in the evaluation of tolerant genotypes (Mutava et al. 2011). In addition to some necessary requirements for use, physiological characteristics (or secondary characteristics) can be used in the selection, because they can enhance accuracy in the identification of superior genotypes in this environment (Araus et al. 2011). Physiological responses to drought tolerance can vary according to the severity and duration of stress imposition, phenological stage, and genetic material (Shao et al. 2008). Regarding phenological stage, maize is particularly very sensitive at flowering stage, according to Edmeades et al. (2000). Drought during this period leads to an increase in the anthesis-silking interval (ASI), which is negatively correlated to yield (Duvick 2005). In the north region of the state of Minas Gerais, in the city of Janau´ba, lack of rainfall limits crop production. During certain times of the year, crop cultivation is only possible with irrigation. For this reason, this region has played an important role in understanding water deficit and the search for tolerant genotypes can favor corn production in these places. It is worth mentioning that plant responses to water stress observed in field conditions are, in general, much more complex than those measured under controlled environmental conditions, because other factors can come into play, in addition to water deficit, influencing the nature of stress response (Lopes et al. 2011). Identification of morphoanatomical modifications, both in root and leaf, has also contributed very much in the genotype selection and in the understanding of tolerance mechanisms in maize under drought conditions (Grzesiak et al. 2010; Kutschera et al. 2010; Zhu et al. 2010). We hypothesized that changes in morphological, anatomical and physiological characteristics favor the survival and higher grain yield of maize genotypes tolerant to drought conditions in the field. Within this context, the objectives of this study were to characterize the morphophysiological and morphoanatomical changes and to evaluate the attributes of grain yield in two contrasting maize hybrids in response to drought stress under field conditions.

Materials and methods Plant material and growth conditions Two maize hybrids with contrasting susceptibility to drought stress were used: DKB 390 (tolerant) and BRS

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1030 (sensitive), the latter being a cultivar bred at Embrapa’s Genetic Enhancement Program (Souza et al. 2013). Both hybrids used in this study are not genetically modified organisms. The resistance and sensitivity of these hybrids were evaluated in previous experiments carried out in Janau´ba, state of Minas Gerais, Brazil. The results showed that the cultivar DKB 390 presented the highest agronomic performance under water deficit, whereas the cultivar BRS 1030 presented the lowest (Martins 2012). The lack of rainfall in Janau´ba limits grain production and the crop cultivation is only possible with irrigation during drought periods. For this reason, this region has played an important role in understanding water deficit in maize crop. The experiment was carried out in 2010 and 2011 under field conditions at Janau´ba experimental station, Minas Gerais, Brazil (altitude of 516 m, 158470 S, 438180 W). Averages of high and low temperatures and relative humidity of air are represented in Fig. 1. Rainfall was practically zero with exceptions for the months of September (0.5 mm) and October (19.7 mm) in 2010 and the months of May (1.3 mm) and October (2.5 mm) in 2011 (data not shown). The experiment was performed in a Red-Yellow Latosol soil type, of medium texture and silty, with the following mineral composition: pH H2O = 6.00, H ? Al cmolc cm-3 = 1.63, phosphorus Mehlich1 mg dm-3 = 34.21, organic matter dag kg-1 = 1.45, Al cmolc dm-3 = 0.05, Ca cmolc dm-3 = 4.02, Mg cmolc dm-3 = 0.95, K mg dm-3 = 265.60, CEC cmolc dm-3 = 7.28, BS % = 77.61, Cu mg dm-3 = 0.82, Fe mg dm-3 = 13.84, Mn mg dm-3 = 16.98, Zn mg dm-3 = 5.30. Basal fertilizing (300 kg ha-1 of the formula 8-28-16) and top dressing (150 kg ha-1 of ammonia sulfate at V4 and 300 kg ha-1 of urea at V8) were carried out according to the soil analysis, following the recommendation for maize in the state of Minas. Plants were watered on a regular basis maintaining optimum moisture in the soil (*-25 kPa), until the stress imposition. All phytosanitary treatments required for the crop were applied. Imposition of water stress and experimental design Soil water content was daily monitored in the mornings and afternoons (9 a.m. and 3 p.m.) with the help of a Watermark soil moisture sensor (tensiometer) model 200SS–5 (IRROMETER, California, USA), installed in the center of the plot in each replication, at the depth of 20 and 50 cm. These sensors detect water tension in the soil based on electric resistance and were attached to digital measurement devices (Watermark meters) from the same company. Water replacement was carried out according to readings obtained with the sensor and water was replaced up to field

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Morphophysiological characteristics

Fig. 1 Maximum and minimum temperatures and relative air humidity during experimental period. a Year of 2010 and b year of 2011

capacity (FC) before the imposition of treatments. These calculations were performed with the help of an electronic spreadsheet, calculated as a function of soil–water retention curve. At pre-flowering, two water treatments were imposed: irrigated and stressed. In the first, water replacement was carried out daily until the soil reached moisture near to FC while there was no water replacement in the second treatment. This stress was kept for 22 days. The experimental design was a randomized block design with four treatments (stressed DKB 390, irrigated DKB 390, stressed BRS 1030, and irrigated BRS 1030) and five replications. The size of the experimental plot was 6 m 9 5.4 m, with six maize rows, spaced 0.90 m apart, totaling an area of 32.4 m2. The two most outside rows were used as border rows, while the two central rows were used for collecting productivity data and the two intermediate rows for the morphophysiological as well as morphoanatomical evaluations collected at the end of the stress imposition. The evaluation of leaf average water potential and morphoanatomical characteristics was performed only in 2010 due to operational problems.

Leaf water potential (midday, Wleaf) was determined at 9 a.m. through a Scholander pressure chamber (Soil Moisture Equipment Corp., Model 3005, Santa Barbara CA, USA) using four fully open leaves in each replication. Leaf senescence characteristic was evaluated through the percentage of dry leaves. The observations and counting in each leaf were visually carried out and a 0–100 % scale was established, where 0 % corresponded to totally green leaves and 100 % to totally senescent leaves (Carlesso et al. 1997). The leaves were counted in eight plants per replication. Chlorophyll relative content (SPAD unit) was determined in the flag leaf using a chlorophyll meter (Model SPAD 502, Minolta, Japan) and ten readings were taken per plant. Total chlorophyll (TC) concentration (lg mL-1), obtained through SPAD unities, was determined through calibration curves for each hybrid. In vivo chlorophyll relative content was determined in leaves with color variations (from yellow to green) followed by the measurement of chlorophyll concentration using Arnon’s method (1949) in those same leaves. Total chlorophyll concentration was determined in 2-mm diameter leaf discs in 10 mL acetone (80 %). Next, the extract was centrifuged at 3,000 g for 10 min and the absorbance of supernatants was evaluated using spectrophotometer at 665 and 645 nm. Leaf area (LA) was estimated by measuring the length (L) and width (W) of all leaves that presented at least 50 % of green area. Leaf area of each leaf was obtained by the equation LA = L 9 W 9 0.75 (Tollenaar 1992). Then leaf area was calculated by adding up the areas of all leaves of the plant. Anthesis-silking interval (ASI) was calculated as a difference, in days, between male and female flowering. Male and female flowerings were calculated as the number of days from seeding until 50 % of the plants of each plot had, respectively, anthesis and visible silks. Leaf stomatal conductance was obtained through the use of a porometer (Decagon Devices, Inc., Pullman, WA, USA). Five readings per leaf per replication were taken between 8 a.m. and 10 a.m. in the flag leaf. Maximum efficiency of photosystem II (Fv/Fm) was determined through a fluorometer (Plant Efficiency Analyser, Hansatech Instruments King’s Lynn, UK) in leaves adapted to dark. Leaf conditioning was carried out with the help of leaf clips with the light intensity in the sensor being 60 % of the equipment’s total capacity, for a period of 5 s at each reading. All readings were also made in the flag leaf, in the morning, between 8 a.m. and 10 a.m.

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Morphoanatomical characteristics For the study of leaf and root anatomy, a sample in the central third part of a fully open leaf below flag leaf and a sample of root (with all regions) were collected in three plants per treatment per replication. Paradermal and transverse sections were performed according to Souza et al. (2009) and Souza et al. (2010) and were photographed under a light-optical microscope (Olympus BX60) attached to a digital camera. The parameters measured in paradermal sections of the leaf abaxial face were stomatal density (number of stomata/mm2) and stomatal functionality (polar diameter/equatorial diameter). Four characteristics were measured regarding leaf blade: abaxial epidermis thickness (BET), number of bulliform cells at every 2 mm (NBC), distance between the vascular bundles (DVB), and mesophyllum thickness (MPT). The evaluation of the leaf blade was performed where there is a higher uniformity of the leaf blade thickness. It started on the fourth vascular bundle with the wider diameter, counting from the midrib region toward the leaf margin. The following parameters were analyzed in root: proportion occupied by the aerenchyma in the cortex (PA), metaxylem cell diameter (XD) and metaxylem number (XN), width of the suberized cell layer present in the hypodermis region (exodermis) (SC), endodermis width (EW), and epidermis width (EPW). Proportion occupied by the aerenchyma in the cortex was calculated by dividing the total aerenchyma area by total cortex area. All these measurements were made with the image processing and analysis program UTHSCSA ImageTool (The University of Texas Health Science Center, San Antonio, TX), using calibrations done with a microscopic ruler photographed with the same magnification used for the photomicrographies. Four measurements of each anatomical characteristic (in each replication) were taken, in the leaf and root.

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Sisvar version 4.3 (Federal University of Lavras, Lavras, Brazil). An index was created to express the tolerance of each hybrid, referred to as SI, where values of each characteristic that were evaluated under stressing condition were divided by values taken under irrigated condition (Souza et al. 2011).

Results Morphophysiological characteristics Regarding leaf water potential (Wleaf), a decrease was observed in all stressed treatments compared to irrigated treatments. However, the sensitive hybrid (BRS 1030) showed a higher decrease in relation to the tolerant (DKB 390) (Fig. 2). As far as percentage of dry leaves is concerned, there were differences only in the year of 2010, when the drought-stressed treatments showed higher percentage of dry leaves (Table 1). It is worth mentioning that the stress index (SI) for the percentage of dry leaves was higher in BRS 1030 subjected to drought stress during the 2 years of evaluation. No differences were observed between the treatments during the 2 years of this study concerning TC concentration, however, BRS 1030 presented higher SI, especially in the year of 2010 (Table 1). DKB 390 subjected to drought showed lower average for LA and irrigated BRS 1030 showed the highest average in 2010 (Table 1). In 2011, irrigated BRS 1030 showed significantly higher results than the other treatments. As for the SI for LA, BRS 1030 presented the highest value in 2010.

Yield components The following data were analyzed at harvest: ear weight (EAW), ear number (EN), ear length (EL), grain yield (GY), 100 grain weight (W100), harvest index (HI) [grain dry weight/(plant dry weight ? grain dry weight) 9 100]. Data analysis Means and standard error (SE) were calculated for all parameters above. For the statistical analysis, the results were submitted to variance analysis (ANAVA) and the means were compared by the Scott-Knott test at 0.05 % significance (P B 0.05), using the statistical program

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Fig. 2 Leaf water potential (Wleaf) during water stress imposition in two drought-contrasting hybrids in the year of 2010. Each bar indicates the treatment average ± SE. Black bar represents the irrigated treatments and the gray bar represents the stressed treatments

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Table 1 Percentage of dry leaves, concentration of total chlorophyll (TC), leaf area (LA), and their respective SI during water stress imposition in two drought-contrasting hybrids (DKB 390 and BRS 1030) Treatments/year

TC (lg mL-1)

Dry leaves (%) 2010

2011

2010

LA (cm2) 2011

2010

2011

Stressed DKB

22.88 ± 2.9a

19.06 ± 1.4a

6.00 ± 4.0a

8.28 ± 3.0a

5,312 ± 1,269c

6,488 ± 900b

Stressed BRS

23.68 ± 1.1a*

19.98 ± 3.6a

9.28 ± 2.8a

11.00 ± 2.3a

7,081 ± 1,300b

7,701 ± 950b

Irrigated DKB Irrigated BRS

13.51 ± 2.5b 12.52 ± 3.0b

15.96 ± 5.0a 11.60 ± 4.0a

9.05 ± 3.6a 12.59 ± 5.5a

8.59 ± 3.0a 11.31 ± 4.0a

7,198 ± 800b 8,370 ± 364a

7,071 ± 410b 8,544 ± 221a

SI DKB

1.69

1.50

0.66

0.94

0.74

0.91

SI BRS

1.89

1.72

0.73

0.95

0.83

0.90

Each value indicates the treatment average ± SE SI stress index (stressed/irrigated) * Means followed by the same letters for the treatments do not differ according to Skott-Knott test at 5 % significance (P B 0.05) Table 2 Anthesis-silking interval (ASI), stomatal conductance (gs), maximum efficiency of photosystem II (Fv/Fm), and their respective SI during water stress imposition in two drought-contrasting hybrids (DKB 390 and BRS 1030) Treatments/year

gs (mmol m-2 s-1)

ASI 2010

2011

2010

Fv/Fm 2011

2010

2011

Stressed DKB

2.50 ± 0.8b

2.75 ± 1.0a

44.5 ± 5.0b

67.0 ± 30b

0.74 ± 0.4a

0.74 ± 1.4a

Stressed BRS

4.25 ± 0.5a*

3.00 ± 0.6a

21.1 ± 1.8c

50.0 ± 23b

0.63 ± 0.3b

0.70 ± 1.6a

Irrigated DKB Irrigated BRS

1.50 ± 0.5c 1.75 ± 1.0c

1.50 ± 0.8b 1.50 ± 05b

198 ± 16a 235 ± 25a

205 ± 25a 210 ± 40 a

0.77 ± 0.2a 0.78 ± 0.3a

0.79 ± 5.0a 0.79 ± 4.0a

SI DKB

1.69

1.50

0.26

0.33

0.96

0.90

SI BRS

1.89

1.72

0.05

0.25

0.80

0.85

Each value indicates the treatment average ± SE SI stress index (stressed/irrigated) * Means followed by the same letters for the treatments do not differ according to Skott-Knott test at 5 % significance (P B 0.05)

Water stress significantly affected the anthesis-silking interval (ASI) in 2010 and 2011 (Table 2). The highest ASI was observed in BRS 1030 in the first year of evaluation. DKB 390, on the other hand, presented the lowest SI in the first and second year of evaluation. In both agricultural years, the highest stomatal conductance (gs) was found in the irrigated treatments. Considering the stressed treatments, BRS 1030 presented the lowest gs in 2010 (Table 2). DKB 390 presented higher SI in both years. BRS 1030 subjected to stress showed the lowest maximum efficiency of photosystem II (Fv/Fm) in the year of 2010 and 2011, even though no statistically significant differences were found between the treatments (Table 2). As for SI evaluation in 2010 and 2011, DKB 390 also showed the highest value. Morphoanatomical characteristics Regarding the analysis of leaf anatomy, DKB 390 subjected to water stress presented the highest stomatal density

(SD), as well as the highest SI (Fig. 3a). Stomatal functionality (SF) increased with water stress in both hybrids. However, DKB 390 presented higher mean in relation to BRS 1030 and higher SI (Fig. 3b). For the abaxial epidermis thickness (BET) (Fig. 3c) and NBC at every 2 mm (Fig. 3d) no significant difference could be found between the treatments or between the SIs (Fig. 3c). DKB 390 subjected to stress was the only treatment that presented a significantly reduced distance DVB and comparing the SI, DKB 390 presented a lower value (Fig. 3e). There was an increase in the MPT with water stress in both hybrids. However, they did not differ among themselves (Fig. 3f). In relation to SI, there was a discrete decrease of MPT in DKB 390. In the analysis of root anatomy, the proportion occupied by the PA significantly increased in both hybrids under water stress, being more noticeable in the DKB 390 subjected to stress (Fig. 4a). XN was higher in both irrigated and stressed DKB 390 when compared to BRS 1030. There was no difference between the SI (Fig. 4b). As for XD, there was a significant decrease in the stressed DKB 390

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Fig. 3 Leaf anatomical characteristics during water stress imposition in two drought-contrasting hybrids in the year of 2010. Each bar indicates the treatment average ± SE. Asterisk means followed by the same letters in each bar do not differ according to Skott-Knott test at 5 % significance (P B 0.05). Black bar represents the irrigated

treatments, white bar represents the stressed treatments, and gray bar represents the stress index (SI). a stomatal density (SD), b stomatal functionality (SF), c abaxial epidermis thickness (BET), d number of bulliform cells at every 2 mm (NBC), e distance between the vascular bundles (DVB), and f mesophyll thickness (MPT)

and the SI was lower in this same hybrid (Fig. 4c). Regarding the width of the suberized cell layer, present in the hypodermis region (exodermis) (SC), it was observed that both hybrids presented higher values in the stressed treatment, being more noticeable in DKB 390 (Fig. 4d). DKB 390 also presented higher SI for SC. No significant difference was found between the treatments or between the SIs in the EW (Fig. 4e). Regarding the EPW, the stressed BRS 1030 showed a significantly higher increase

than the other treatments. A higher SI was also observed in BRS 1030 regarding EPW (Fig. 4f).

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Yield components A decrease of EAW was observed among the stressed and watered treatments in the 2 years of study and DKB 390 presented higher SI (Table 3). Following the same pattern as EAW in 2010, there was a decrease of the EN in the

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Fig. 4 Root anatomical characteristics during water stress imposition in two drought-contrasting hybrids in the year of 2010. Each bar indicates the treatment average ± SE. Asterisk means followed by the same letters in each bar do not differ according to Skott-Knott test at 5 % significance (P B 0.05). Black bar represents the irrigated treatments, white bar represents the stressed treatments, and gray bar

represents the stress index (SI). a proportion occupied by the aerenchyma in the cortex (PA), b metaxylem number (XN), c metaxylem diameter (XD), d width of the suberized cell layer, present in the hypodermis region (exodermis) (SC), e endodermis width (EW), and f epidermis width (EPW)

stressed treatments and an increase of SI in DKB 390 (Table 3). There was no difference in the EL between the treatments in 2010 and 2011 and the SI of DKB 390 was slightly higher in 2010 (Table 3). Table 4 shows that the stress in the first year of evaluation (2010) led to lower GY than in the second year (2011). In both years, the stressed BRS 1030 presented lower GY and lower SI than the stressed DKB 390 (Table 4).

In the evaluation of W100, in 2010, the values of the stressed treatments were lower than those watered treatments. No difference was found in 2011 or in the evaluation of SI (Table 4). For HI, the stressed BRS 1030 showed a lower value in 2010 and there was no difference between the treatments in 2011 (Table 4). Concerning the SI for HI, DKB 390 presented the highest value in both years, even though the difference was less pronounced in the second year.

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Table 3 Ear weight (EAW), ear number (EN), ear length (EL), and their respective SI during water stress imposition in two drought-contrasting hybrids (DKB 390 and BRS 1030) Treatments/year

EAW (g)

EN

2010

2011

EL (cm)

2010

2011

2010

2011

Stressed DKB

3.489 ± 520b

3.935 ± 15b

31.84 ± 3.3b

20.25 ± 5a

13 ± 7a

12 ± 5a

Stressed BRS

2.878 ± 566b*

3.340 ± 10b

27.66 ± 2.8b

21.75 ± 4a

12 ± 4a

12 ± 6a

Irrigated DKB Irrigated BRS

9.752 ± 415a 8.497 ± 375a

4.750 ± 20a 4.165 ± 22a

47.16 ± 4.7a 49.50 ± 6.0a

22.00 ± 5a 23.75 ± 2a

18 ± 4a 18 ± 4a

15 ± 5a 17 ± 3a

SI DKB

0.41

0.83

0.69

0.91

0.72

0.70

SI BRS

0.34

0.81

0.56

0.90

0.69

0.70

Each value indicates the treatment average ± SE SI stress index (stressed/irrigated) * Means followed by the same letters for the treatments do not differ according to Skott-Knott test at 5 % significance (P B 0.05)

Table 4 Grain yield (GY), 100Grain weight (W100), Harvest index (HI), and their respective SI during water stress imposition in two droughtcontrasting hybrids (DKB 390 and BRS 1030) Treatments/year

GY (kg ha-1) 2010

W100 (g) 2011

2010

HI 2011

2010

2011

Stressed DKB

2.963 ± 230b

7.063 ± 149b

32.12 ± 0.8b

40.70 ± 02a

0.30 ± 0.03b

0.37 ± 0.1a

Stressed BRS

2.204 ± 110c*

5.980 ± 113c

32.47 ± 0.5b

37.60 ± 01a

0.20 ± 0.01c

0.35 ± 0.1a

Irrigated DKB Irrigated BRS

7.674 ± 209a 6.725 ± 410a

9.600 ± 750a 8.962 ± 160a

36.01 ± 0.5a 36.54 ± 0.2a

40.80 ± 01a 39.21 ± 0.4a

0.43 ± 0.05a 0.40 ± 0.03a

0.44 ± 0.09a 0.40 ± 0.05a

SI DKB

0.42

0.74

0.89

0.89

0.69

0.82

SI BRS

0.33

0.66

0.89

0.88

0.50

0.80

Each value indicates the treatment average ± SE SI stress index (stressed/irrigated) * Means followed by the same letters for the treatments do not differ according to Skott-Knott test at 5 % significance (P B 0.05)

Discussion It can be observed that in Janau´ba, MG experimental station there are more favorable conditions for conducting drought experiments in relation to other regions in the state, due to rainfall absence (or low rainfall) and high temperatures (Fig. 1). Comparing the characteristics that were evaluated in the 2 years of study, it is clear that there was a higher effect of water stress in 2010 than in 2011. This could be due to the higher temperatures in 2010 (Fig. 1), which added to drought conditions, and could have led to a higher impact over the hybrids, especially the sensitive hybrid (BRS 1030). Pradhan et al. (2012) working with wheat, also reported a greater effect on physiological and production characteristics, when water and high temperatures stresses were combined. They observed that one of the genotypes studied was more tolerant than the other. Upon evaluation of Wleaf, it was observed that water stress led to lower values in both hybrids. However, DKB 390 (tolerant) presented higher water status than its contrasting counterpart BRS 1030. Under field conditions,

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Vitale et al. (2007) also observed a decrease of Wleaf in maize subjected to drought. This higher maintenance of water status in DKB 390 cannot be due to stomatal closure because it showed higher gs. These two characteristics (gs and Wleaf) are intimately linked because a higher water status can favor a higher stomatal conductance, leading to CO2 flow and to leaf cooling by transpiration. According to Jones et al. (2009) and Mutava et al. (2011), droughtresistant genotypes with high yield have been identified by canopy cooling and to escape from drought they need to consume more water. Thus, DKB 390 with a higher water status can release more water through the stomata openings, which in turn promotes a higher canopy cooling. Leaf senescence is one of the first visible symptoms to be observed in drought stress (Smit and Singels 2006). The hybrid DKB 390 presented lower SI for percentage of leaf senescence. Despite the absence of significant differences for the TC concentration between the treatments, a higher value of SI was observed in BRS 1030. This leaf senescence can be caused by a higher degradation of pigments by oxygen reactive species (ORS), which, under water

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deficit condition, has its production of ORS increased (Karuppanapandian et al. 2011). Resistant hybrids tend to have a higher antioxidant system (Moussa and Abdel-Aziz 2008), which can be the cause of lower senescence in DKB 390. Smaller LA found in DKB 390 can decrease leaf transpiration surface, helping in the plant survival, by maintenance and control of water use when facing water stress (Shao et al. 2008). Anthesis-silking interval (ASI) has been one of the major secondary characteristics used to identify maize genotypes in genetic enhancement programs aiming at drought tolerance (Araus et al. 2011; Badu-Apraku et al. 2011; Hao et al. 2011). Definitely, BRS 1030 showed higher lack of synchronism between the inflorescences in 2010. Therefore, this can be one of the factors that could have affected yield in this hybrid (Dubey et al. 2010). Still considering ASI in the year when the temperature was higher (2010), BRS 1030 could have presented higher interval due to a higher sensitivity in hotter days, as there are reports showing that temperatures over 33 °C can delay or even inhibit flowering events (Edreira et al. 2011). A significant decrease in the Fv/Fm relationship was verified in BRS 1030. With the increase of water deficit, leaves wilt in response to stomatal closure, photosynthesis decreases, and photochemical activity is lost (damage in the photosystem) due to an excess of energy. Thus, the Fv/ Fm relationship is one of the major parameters used to evaluate damage in photosynthesis system, since maximum efficiency of photosystem II indicates when all reaction centers are open (Baker and Rosenqvist 2004). One of the factors that can be facilitating a higher stomatal conductance in DKB 390 is the higher SF and SD found in leaves. Stomatal closure is one of the most noticeable responses to drought, leading to a decrease in gas exchange (Farooq et al. 2009). However, a higher number of stomata can favor higher gas input, which decreases stomatal resistance. Ennajeh et al. (2010) observed the same behavior in olive trees with higher SD in drought-resistant cultivars. A higher stomatal functionality can increase water use efficiency, since this parameter is related to a smaller area of stomatal opening (polar and equatorial diameter of stomata) (Souza et al. 2010). No differences were observed between the treatments for BET. Makbui et al. (2011) found thicker adaxial and abaxial epidermis in soybean plants subjected to drought. In spite of absence of modifications in bulliform cells in this study, this characteristic can be promising in studies of drought tolerance, as it is involved in leaf rolling, which appears to be a mechanism used by the plant to avoid transpiration (Alvarez et al. 2008).

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An important characteristic, such as the small distance DVB of the leaves, was also found in DKB 390 under water restriction and high temperatures. This characteristic provides higher ability in translocating photoassimilates and higher distribution of water in leaves (Sage 2004). Similar data were also found in Phragmites communis subjected to drought (Gong et al. 2011). Water stress led to an increase in MPT in both DKB 390 and BRS 1030. According to Poorter and Bongers (2006), plants submitted to abiotic stresses increase their leaf thickness, because this plasticity leads to an increase of leaf nitrogen allocation, which increases the photosynthesis capacity and the efficient use of nitrogen. A higher proportion occupied by the PA was found in both hybrids under stress treatments compared to irrigated treatments. However, DKB 390 presented the highest increase. Aerenchymas are defined, in general, as a specialized tissue characterized by intercellular spaces filled by gases and its formation, in maize, involves lyses and programmed cell death (Lenochova´ et al. 2009). Aerenchyma formation in maize can be involved with other types of stress tolerance, such as flooding (Souza et al. 2009) and nutrient deficiency (Postma and Lynch 2011). Gowda et al. (2011) also found expressive aerenchyma formation in rice subjected to water deficit. A higher proportion of aerenchyma in DKB 390 can allow enhanced soil exploration and water intake, since these structures decrease the metabolic cost of growing roots, due to a decrease in the number of cells that are undergoing cellular respiration (Zhu et al. 2010). Few studies involving drought tolerance in maize and aerenchyma formation can be found in the literature. Zhu et al. (2010) observed that, in field studies with maize under drought conditions, genotypes with a higher proportion of aerenchyma in their roots presented better performance (higher root growth and higher biomass of the above-ground part). DKB 390 presented a higher number of metaxylem cells and a decrease in the diameter of these cells. These characteristics of DKB 390 can indicate a higher hydraulic conductivity, which increases the ability of transporting water (Li et al. 2009). A smaller diameter of metaxylem cells is related to the decrease of embolism risk and increase in the water flow resistance. As for the vascular bundles, a higher number can increase the probability that the water reaches its destiny or that the flow occurs (Souza et al., 2009). Same modifications in xylem cells were observed in drought-resistant genotypes of maize (Pen˜aValdivia et al. 2005; Li et al. 2009). No differences were observed between the treatments concerning EW. However, an increase was observed in the width of the suberized cell layer, present in the hypodermis region (exodermis) (SC), for the stressed treatments, especially in DKB 390. Both endodermis and exodermis

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have several roles in the roots, but in general, they are layers of cells specialized in selecting or preventing entry of toxic substances or microorganisms. In the case of water stress, these two layers can prevent water from escaping from xylem vessels into the soil, avoiding dehydration (Enstone et al. 2003; Pen˜a-Valdivia et al. 2005). It was observed in this study that the sensitive hybrid (BRS 1030) presented wider root EPW. An explanation for this behavior could be that the epidermis increases its role as barrier, responding as a substitution to a thinner exodermis and endodermis (Souza et al. 2009). The stress effect in yield attributes caused by water deficit is remarkable. Differences between the stressed and irrigated conditions were evident in this study. The results found in DKB 390 for GY in the 2 years of evaluation confirmed its higher tolerance to drought compared to BRS 1030. The increase of HI is one of the reasons that could have led to a higher yield in DKB 390, namely a higher differential allocation of photoassimilates to the ear during its life cycle. This difference of allocation between the hybrids highlights even more the idea that tropical maizes are strongly limited by sink (Borra´s et al. 2004). For the parameters EAW, EM, and W100), a significant decrease was observed in the stressed treatments (mainly in the first year of evaluation), but no differences were observed between the hybrids under the same condition. In several studies, such parameters were also considered relevant factors in drought tolerance (Betra´n et al. 2003; Monneveux et al. 2006; Dubey et al. 2010; Hao et al. 2011). The results of the present study show that in field conditions, under water deficit stress, DKB 390 root presented a higher proportion occupied by the aerenchyma in the cortex, an increase in exodermis width, and a higher amount of metaxylem (with smaller diameter). Higher number of stomata and smaller distance between the vascular bundles in the leaf blade were found in the leaves of this same hybrid. Furthermore, DKB 390 presented smaller percentage of dry leaves, higher synchronization of inflorescences, higher stomatal conductance, and higher Fv/Fm relationship. Drought significantly affected maize hybrids evaluated in this study. DKB 390 presented changes in morphophysiological and morphoanatomical characters, which favored its survival in environments with water deficit, resulting in higher yield. Author contribution Dr. Thiago Correˆa de Souza carried out the experiment and wrote the article. Dr. Paulo Emı´lio Pereira de Albuquerque helped monitoring the soil moisture and its interpretations. MSc Leandro de Oliveira Lino and Elı´cia Trindade Alves contributed with morphophysiological and morphoanatomy analysis. Drs. Paulo Ce´sar Magalha˜es and Evaristo Mauro de Castro were

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responsible by yield components, morphoanatomy analysis, and as well as its interpretations. All the authors read and approved the final manuscript. Acknowledgments The authors would like to thank Capes for the scholarship; Centro Nacional de Pesquisa de Milho e Sorgo, Fundac¸a˜o de Amparo a` Pesquisa do Estado de Minas Gerais (FAPEMIG), and Laborato´rio de Anatomia Vegetal da Universidade Federal de Lavras for providing the facilities and materials that made this research possible.

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