Upland rice: phenotypic diversity for drought tolerance

Upland rice is cultivated mostly in Latin America and Africa by small farmers and in areas with risk of dry spells. This study evaluated morphophysiological mechanisms of upland rice associated to drought adaptation. A set of 25 upland rice genotypes were grown in a plant phenotyping platform during 2015 and 2017 under regular irrigation and water restriction. We evaluated morphophysiological traits in shoots (vegetative structures growth, gas exchange, water use efficiency, carboxylation efficiency, water status) and roots (length, surface area, volume and diameter), as well as agronomic traits (grain yield and its components). There was a reduction in grain yield by up to 54 % and 58 % in 2015 and 2017, respectively, under water deficit. Five upland rice genotypes with the best yield performances in both water treatments applied were recommended to the upland rice-breeding program: Bico Ganga, BRS Esmeralda, BRSMG Curinga, Guarani, and Rabo de Burro. In this study, morphophysiological traits associated to drought tolerance concerned the plant high capacity to save water in the leaves, low leaf water potential, high ability to reduce vegetative structures, high water use efficiency, high photosynthetic capacity, and improved capacity to absorb water from drying soil, either by osmotic adjustment or additional investment into the root system. Therefore, we concluded that different secondary traits contributed to drought tolerance and should be evaluated along with grain yield to improve efficiency of breeding selection.


Introduction
Rice (Oryza sativa L.) is essential for food security for more than half of the world's population (Jumaa et al., 2019). Since rice has an evolutionary peculiarity of semiaquatic, flooded rice paddies have become the major form of cultivation, growing in irrigated and rainfed lowland conditions, equivalent to 75 % and 19 % of the global production area, respectively (Kikuta et al., 2016). Increasing grain yield of irrigated areas is not enough to supply future demand for rice; furthermore, expansion of production areas is restricted, due to the water scarcity (Parthasarathi et al., 2012). Upland rice represents 4 % of the global rice production and is grown less than 9 % of total rice acreage in Asia, 46 % in Latin America, and 47 % in West Africa (Kikuta et al., 2016). According to Singh et al. (2014), upland rice accounts for 84 % of the total area in Sub-Saharan Africa and it is cultivated mostly by smallholder farms with an average area smaller than 0.5 ha. On the other hand, in Latin America, upland rice is cultivated in large-areas of mechanized harvesting (Bernier et al., 2008).
Drought is one of the most severe abiotic stresses limiting rice yield worldwide and poses a serious threat to rice sustainability in rainfed agriculture (Wu and Cheng, 2014). According to Heinemann et al. (2015), rice yield in upland cultivation (tropical regions mainly) has its yield potential reduced by up to 35 % due to drought-stress conditions.
Reduction in water availability for plants results in a complex response characterized by a decrease in the water potential of its tissues, leading to several changes in different plant processes (Rosales et al., 2012). Some processes reported for upland rice are (a) appropriate phenological patterns that combine crop growth and the amount of water available in the soil (water environment), (b) deep root system, (c) thick stems and reduced number of stomata, (d) osmotic adjustment to maintain cell homeostasis and, consequently, avoid a rapid decrease in leaf-water potential, and (e) senescence delay, also known as stay-green trait, which allows the maintenance of the photosynthetic capacity and the photoassimilate remobilization for a longer time period (Fukai and Cooper, 1995;Boonjung and Fukai, 1996).
Thus, the establishment of sustainable crop systems of upland rice requires better understand of changes in the morphophysiological mechanisms, contributing to drought tolerance and yield effects. This study aimed to (a) identify a series of morphophysiological and agronomic traits related to drought tolerance in upland rice genotypes of Embrapa Core Collection under greenhouse cultivation, and (b) characterize morphophysiological components to be used as indicators for drought tolerance for plant breeding processes.

Experimental conditions
The experiments were carried out under a greenhouse condition at the plant phenotyping platform facility the Integrated System for Drought-Induced Treatment (Portuguese acronym SITIS) from Feb to June 2015, and from Aug 2017 to Jan 2018, 16°28'00" S, 49°17'00" W, altitude of 823 m. At the facility, 384 soil columns (diameter: 25 cm; height: 100 cm) were placed on a digital scale to monitor the water amount in each column. The soil, characterized as red latosol (Oxisol), was sieved through 125 mm mesh to remove larger aggregates and it was enriched with minerals, including 1.125 g kg -1 of 5-30-15 formulation, and 0.250 g kg -1 of ammonium sulfate after germination. Urea was applied at the beginning of tillering (V4-V5 stage; 0.350 g kg -1 ) and in the panicle differentiation (R1 stage; 0.150 g kg -1 ), four days before the period of water restriction.
The treatments consisted of combinations of two water levels including normal watering (control treatment) and restriction water (stress treatment) conditions. In the control treatment, the amount of soil water was equivalent to 80 % -85 % of field capacity (FC) established and kept throughout the crop cycle. For the stress treatment, irrigation was performed until the plant reached the reproductive stage (R2/R3), followed by suspension of irrigation for five days, with subsequent replacement of only 50 % of evapotranspirated water at the plate placed on the column bottom for 10 days. The amount of evapotranspirated water was estimated based on the water quantity required to keep soil FC at 80 % -85 % in the control treatment. Water stress was kept until the control plants reached R6 (grain depth expansion) / R7 (grain dry down) stage. After this period, irrigation was restored until the end of the crop cycle, R8 (at least one grain on the main stem panicle with a brown hull) / R9 (all grains that reached R6 have brown hulls). In the control columns, the evapotranspiration rate was determined daily (difference between the reference mass and the column/day mass) and restored through irrigation placed on the soil surface to achieve the initial mass (reference mass) again. Each column contained three plants.

Grain yield and yield components
The agronomic traits evaluated were grain yield (GY -g column -1 , which means the total mass of grains, in grams, obtained for three plants per column) and its components, such as the number of filled grains (NFG, filled grains average in six panicle column -1 ), number of empty grains (NEG, empty grains average in six panicle column -1 ), and 100-grain mass (100GM, g). The last variable was evaluated in 2015. Spikelet sterility was estimated as SS = (NEG × 100) TG -1 , where SS = spikelet sterility, NEG = number of empty grains, and TG = total number of grains.

Shoot growth
The following assessments were made for shoot (vegetative structures) growth and reproductive organs traits: (a) leaf area (LA, cm 2 ), average of two flag-leaf of two plants in column, using LI-COR leaf area meter; (b) plant height (PH, cm); (c) tiller number (TN, units); (d) panicle length (PL, cm); (e) shoot dry matter biomass (SDMB, g), through drying samples at 65 °C until a constant weight was achieved and (f) panicle number (PN, units). Data on PH, TN, SDMB, and PN were the average of three plants in the column. Additionally, LA and TN were measured on the last day of water restriction. The PH, PL, SDMB, and PN were obtained at harvesting time. LA and PL were measured in 2015.

Root phenotyping
The root system was evaluated according to the methodology described by Lanna et al. (2016). Briefly, to carry out the root system capture, acrylic tubes were installed inside the columns and three rice plants were planted around the tube. The root system growth was assessed by measuring length (cm), surface area (cm 2 ), volume (cm 3 ) and diameter (mm) of the roots through images generated by CI -600 root scanner, with quantification by the WinRhizo software. Root images corresponding to depth 1 (5 to 25 cm) and 2 (25 to 45 cm) were taken on the 1 st day after irrigation cut-off (phase I), 5 th day after irrigation cut-off (phase II) and 10 th day after the plants received 50 % of water at the column base (phase III). These parameters were evaluated in 2017.

Gas exchange
Gas exchange rates were taken on flag leaves of two plants in each column and measurements were made using a portable gas exchange analyzer in the infrared region (LCpro+). The parameters measured were: photosynthetic rate (A, μmol CO 2 m -2 s -1 ), transpiration rate (E, mmol H 2 O m -2 s -1 ), stomatal conductance (gs, mol H 2 O m -2 s -1 ), and internal CO 2 concentration (Ci, μmol mol -1 ). The equipment was set to use temperature and concentrations of 370 -400 mol mol −1 CO 2 in the air, the reference condition used in the IRGA phothosynthesis chamber. The photon flux density photosynthetic active (PPFD) used was 1200 μmol [quanta] m −2 s −1 . The minimum equilibration time set for performing the reading was 2 min. Measurements in both control and stressed plants were carried out at from 07h30 to 11h00 a.m. on three evaluation dates during the water deficit period. These dates included the 1 st day after irrigation cut-off (phase I), 5 th day after irrigation cut-off (phase II), and 10 th day after the plants received 50 % of water at the column base (phase III). Water use efficiency (WUE, μmol CO 2 mol -1 H 2 O) was calculated as the ratio between A and gs (Rosales et al., 2012). Carboxylation efficiency (CE, (μmol m -2 s -1 ) (μmol mol -1 ) -1 ) was expressed as the ratio between A and Ci (Silva et al., 2013). Upland rice responses to drought Sci. Agric. v.78, n.5, e20190338, 2021

Water status
Leaf water potential (Ψw) was evaluated between 05h00 and 06h00 a.m. using a Scholander pressure chamber (Scholander et al., 1965). The reading was determined at the extremity (tip) of two flag leaves of the primary tiller of two upland rice plants at the end of the water restriction period. Pressure was applied until exudation from the cut made in the leaf collar. Leaf relative water content (RWC, %), osmotic potential (Ψs, MPa), and osmotic adjustment (OA; MPa) were also determined according to the methodology described by Bajji et al., 2001. These parameters were evaluated in 2015.

Experimental design and statistical analysis
All 25 genotypes were evaluated in a 5 × 5 lattice design with 12 repetitions: six repetitions (columns) were for irrigated conditions, and other six repetitions were used for the water deficit treatment, totaling 300 experimental units (with each column containing three plants). Among the six repetitions per water treatment, three repetitions were used for destructive (LA, Ψw, RWC, Ψs and OA; only in 2015) and three for non-destructive measurements (gas exchange, shoot structure, grain yield and its components). For all measurements of shoot traits, transformation √x + 1.0 was applied (where x represents the analyzed variables), which is often used for measurable or count data for normalizing and reducing data skewness (Shapiro and Wilk, 1965, normality test 5 %). The transformed data were subjected to the analysis of variance (ANOVA) based on a fixed linear model and to the joint analysis within each year (2015 and 2017), considering the following: blocks effects, two water levels effects, 25 genotypes, and water level × genotype interaction. The treatment means were compared by the Scott-Knott test (p < 0.05), due to a large number of treatments used in this study, which facilitated the ranking of 25 genotypes into homogeneous groups, without ambiguity. These analyses were carried out using the R platform (R Core Team, 2018). For the root traits, the data were analyzed by the GENES statistical analysis software. The joint analysis of variance was performed between the environments (irrigated and stressed) for each depth, and the significant differences were tested by the Tukey test at p < 0.05.

Results and Discussion
In crops, such as upland rice, where seeds are the product of interest, the main criteria for selecting agronomical tolerance to drought are the traits that lead to higher grain yield. In this study, the analysis of grain yield showed a significant difference (p < 0.05) for all variation sources. For 2015 and 2017, the genotypes accounted for 41 % and 50 % of the total sum of squares, while the environment (water level) accounted for 41 % and 44 % and the genotype versus environment interaction accounted for 18 % and 7 %, respectively. The agronomic performance (grain yield) of genotypes cultivated under two water treatments in 2015 and 2017 is shown in Figures 1A and 1B. In 2015, Bico Ganga, BRS Esmeralda, BRSGO Serra Dourada, BRSMG Curinga, Casca Branca, Guarani, Rabo de Burro, Rio Doce, and Três Meses Branco showed better yield under drought (average grain yield 70.9 g column -1 ) and irrigated (average grain yield 119.8 g column -1 ) conditions. In 2017, Agulhão, Bico Ganga, BRS Esmeralda, BRS Primavera, BRS Soberana, BRSMG Curinga, Guarani, and Rabo de Burro were more productive under drought (average grain yield 40.90 g column -1 ) and irrigated (average grain yield 63.98 g column -1 ) conditions. Among the upland rice genotypes evaluated, Bico Ganga, BRS Esmeralda, BRSMG Curinga, Guarani, and Rabo de Burro showed better agronomic performance at both water levels in both two years of trials and were then ranked as top genotypes. These genotypes probably present favorable alleles of drought tolerance that may be useful in breeding programs of upland rice. Two of these genotypes are modern cultivars (BRS Esmeralda and BRSMG Curinga), which could be qualified as parents in breeding programs of upland rice. For yield components, the average number of filled-grains and empty-grains was 138 and 46 in 2015, and 266 and 82 in 2017, respectively, in rice cultivated under irrigated condition (Table 2) In both years of trials, environmental conditions of phenotyping platform SITIS were severe. Particularly in 2017, in addition to artificially imposed water stress, the maximum temperature of 44.7 °C was 6.7 °C higher than the conditions of the 2015 trial, during the water deficit period. In addition, the minimum relative humidity of 26 % was 42 % lower than that of the 2015 trial (Table  3). According to Choudhary et al. (2018), drought commonly occurs combined with other environmental stresses, such as excessive light incidence, heat, and low relative humidity, and characterizes multiplicity of stresses in the tropics. For rice, along with drought, high temperature (up to 33.5 °C) contributed to yield reduction due to the shortening of the vegetative period and high spikelet sterility (Peng et al., 1995;Matsui et al., 1997;Shah et al., 2011). According to Bernier et al. (2008), practices based on the assessment of agronomic performance of crop species require a long procedure, which limits breeding efficiency. Thus, a better understand of mechanisms of drought tolerance is necessary, since the association between main (grain yield and its components) and secondary (morphophysiology) traits could provide greater selection efficiency. For this, identifying morphophysiological traits related to drought tolerance is relevant to assist in the identification of mechanisms underlying these adaptation processes and thus in the selection of tolerant genotypes. In this study, upland rice plants reacted to drought stress by slowing down their growth (  Fischer et al. (2003) and Chaves et al. (2009), reduction of leaf growth and stem elongation in rice plants are the first processes affected by drought and could be considered as a tolerance mechanism, since they reduce the transpiration capacity, and consequently, plant demand for water. Furthermore, slowed growth (due to reduction of stomatal conductance, CO 2 assimilation and, consequently, photoassimilates production and accumulation) has been suggested as an adaptive trait for plant survival under stress. This trait allows plants to divert assimilates and energy into protective molecules to deal with stress (Zhu, 2002) and/or keep root growth by increasing water acquisition (Chaves et al., 2003;Pandey and Shukla, 2015).
The effect of the drought treatment was also evaluated by characterization of the root system, an important organ to increase rice yield under water stress (Pandey and Shukla, 2015;Kundur et al., 2015). According to Kato et al. (2006), rice root is complex, combining various root morphologies and showing considerable genotypic variation, also subjected to environmental effects. Thereby, a deep root system could improve adaptation of upland rice during drought by increasing capacity of extraction water, keeping high leaf water status with better crop performance under drought conditions (Kamoshita et al., 2004;Mishra and Salokhe, 2011). In this study, the analysis of the root system of upland rice showed a significant difference (p < 0.05) for most variation sources. At depth 1 (5 -25 cm), the genotypes accounted for 14 % of the total sum of squares, the environment (water level) accounted for 54 %, and the double interaction, genotype versus water lever, accounted for 32 %. At depth 2 (25 -45 cm), the genotypes accounted for 62 % of the total sum of the square, the environment (water level) accounted for 20 %, and the double interaction, genotype versus water level, accounted for 17 %.
The root system properties (length, surface area, volume, and diameter) of upland rice plants during the drought period are shown in Figure 2. Under irrigated condition, the genotypes that stood out mostly in terms of length, area, volume, and root diameter were IRAT 112, Agulhão, BRSMG Curinga (top genotype), Comum, Rabo de Burro (top genotype), and Saia Velha, at both depths. Under drought condition, the highlight was BRSMG Curinga followed by Agulhão, Comum, Rabo de Burro, and Saia Velha. Therefore, among top genotypes, BRSMG Curinga, and Rabo de Burro presented greater robustness of the root system, mainly at depth 2 (25 -45 cm), irrespective of the water level applied. This is in accordance with Pandey and Shukla (2015), which describe that under water deficit, root growth is usually kept, while shoot growth is inhibited. Conversely, Ji et  Capital letters compare the genotypes within each water regime and small letters the water levels within each genotype. Means followed by the same capital letter in the column and means followed by the same letter on the lines not differ by the Scott-Knott test 5% error probability. Transformed data in square root of Y + 1.0 -SQRT (Y + 1.0) to statistical analysis. Upland rice responses to drought Sci. Agric. v.78, n.5, e20190338, 2021 al. (2012) found a more extensive deeper root growth in a tolerant rice cultivar, IRAT109, after 20 days of irrigation cut-off. The findings of our study indicate a mechanism at the molecular level underlying a constitutive root growth for the root traits evaluated. Water deficit is an important environmental constraint and influences all physiological processes in plant growth, affecting gas exchange mechanisms (Ma et al., 2018). The stress effects on A, E, gs, Ci, WUE, and CE in upland rice plants are shown in Table 5. During phase I, where control and stress columns were in similar conditions of soil water availability, there was genetic variability among rice accessions, implying a contrast for the gas exchange traits evaluated, in both years of    When water deficits start to increase, leaf stomatal conductance usually decreases faster than carbon assimilation, leading to increased WUE. The WUE reflects the multiple environmental stimuli perceived and the capacity of a particular genotype to sense the onset of changes in moisture availability and therefore to fine-tune its water status in response to the environment (Wilkinson, 2004;Blankenagel et al., 2018).
However, despite the negative impact of water deficit on gas exchange, in both years of trials, Bico Ganga, BRS Esmeralda, BRSMG Curinga, and Guarani (top genotypes) improved their WUE (44 %) when compared with optimal irrigation conditions. This was most probably due to higher stomatal control efficiency, keeping approximately 40 % of the photosynthetic process and drastically reducing stomatal conductance (70 %) by closing the stomata process. Although Rabo de Burro did not show increase in WUE, it presented a recovery of the gas exchange apparatus compared to irrigated plants, which can be justified partly by its vigorous root system.
In addition to increased relative stomatal limitation, drought stress is responsible for reducing maximum Rubisco carboxylation activity and electron transport and therefore ribulose bisphosphate (RuBP) regeneration (Perdomo et al., 2017). The carboxylation efficiency could be considered an estimate of the Rubisco activity, illustrating its limitations under stress conditions Upland rice responses to drought Sci. Agric. v.78, n.5, e20190338, 2021 (Niinemets et al., 2009). In our study, all upland rice genotypes showed a poor capacity to overcome limitation in CO 2 diffusion by stomata and mesophyll and effective CO 2 fixation (70 % of CE reduction) during phase II for both years of trials. After replenishing 50 % of water at the column base for 10 days, recovery of 55 % and 64 % in the carboxylation efficiency was observed in 2015 and 2017, respectively. Considerable loss of Rubisco activity during stress conditions were also reported for sugarcane subjected to water deficit (Saliendra et al., 1996;Vu and Allen Jr., 2009). Overall, a response pattern was not observed among genotypes with greater yield performance under water deficit, since they showed divergent physiological responses of gas exchange.
Furthermore, remobilization of photoassimilates from vegetative into reproductive structures may have a significant effect on grain yield, although this component was not evaluated in our study. As demonstrated for cereals (Blum et al., 1994) and legumes (Chaves et al., 2002), nutrient pre-anthesis reserves are used for grain filling in addition to current assimilates. In rice, droughtinduced leaf senescence also promotes assimilate allocation to grains under development, shortening grain filling and increasing the grain filling rate (Sehgal et al., 2018). Moreover, senescence and reserve mobilization are integral components of plant development and basic strategies in stress mitigation (Lemoine et al., 2013).
Water stress effects on Ψw, RWC, Ψs, and OA, evaluated only in the 2015 trial, are shown in Table 6. Among top genotypes, BRS Esmeralda, BRSMG Curinga, Guarani, and Rabo de Burro showed a more pronounced gradient of ψ w and probably enhanced water absorption capacity. Besides, advance of the most severe internal damage may have reduced in the reproductive organs under the drought period. Conversely, Bico Ganga kept high water potential during the water deficit period, which may be associated to a more robust root system in the second soil layer and thus higher panicle water potential, which probably contributes to increased grain yield. According to Guimarães et al. (2016), plants that prevent dehydration presented higher water potential and earliness in flowering, lower height, lower leaf area or lower tillering. Regarding the trait RWC, which is directly related to the plant water status, values ranged from ~ 82 % in leaves under irrigated condition to 75 % for stressed plants. On the other hand, BRSMG Curinga Capital letters compare genotypes within each water regime and small letters compare water regimes within each genotype. Means followed by the same capital letter in the column and means followed by the same letter on the rows do not differ by the Scott-Knott test 5 % error probability. Transformed data in square root of Y + 1.0 -SQRT (Y + 1.0) for the statistical analysis.