Sorghum [Sorghum bicolor (L.) Moench] is an important cereal

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1 Research Is the Stay-Green Trait in Sorghum a Result of Transpiration Sensitivity to Either Soil Drying or Vapor Pressure Deficit? Sunita Choudhary, Raymond N. Mutava, Avat Shekoofa, Thomas R. Sinclair,* and P. V. Vara Prasad Abstract Persistence of green leaves during seed fill, referred to as a stay-green trait, has been investigated in sorghum [Sorghum bicolor (L.) Moench] as an approach to increasing yields under water-limited conditions. An hypothesis to explain the observation of stay green in some sorghum genotypes and not in others is that the genotypes expressing the trait employ mechanisms to increase availability of soil water during seed fill. In this study, the expression of two mechanisms resulting in soil water conservation to allow greater water availability during seed fill was explored among 12 sorghum genotypes. One mechanism is an earlier decrease in transpiration with soil drying so that the rate of soil water loss is decreased earlier in the soil drying cycle. The second mechanism is a limitation on transpiration rate at high vapor pressure deficit (VPD) so that soil water is conserved on days when midday VPD is high. Field studies were undertaken to identify seven genotypes that consistently expressed the stay-green trait and five genotypes that did not exhibit this trait. The range of the threshold for the decrease in transpiration rate with soil drying was similar among the two sets of genotypes. Similarly, the expression of the limited transpiration rate under high VPD was found for both sets of genotypes. There was no evidence in these studies that the stay-green trait was closely linked with either mechanism of water conservation. S. Choudhary, A. Shekoofa, and T.R. Sinclair, Crop Science Dep., North Carolina State Univ., Raleigh, NC ; R.N. Mutava and P.V. Vara Prasad, Dep. of Agronomy, Kansas State Univ., Manhattan, KS Received 21 Jan *Corresponding author (trsincla@ ncsu.edu). Abbreviations: FTSW, fraction of transpirable soil water; NTR, normalized transpiration ratio; PVC, polyvinyl chloride; TR, transpiration ratio; VPD, vapor pressure deficit. Sorghum [Sorghum bicolor (L.) Moench] is an important cereal crop that is grown globally for food and feed. The United States is the largest producer and fourth-largest sorghum-cultivating country, but more than 90% of the world s sorghum area lies in the arid and semiarid areas, mainly in Africa and Asia (FAO website: accessed 19 June 2013). Sorghum is the most important cereal in semiarid regions, and its area of production is consistently increasing in eastern and western Africa (Deb and Bantilan, 2003). A concern is the declining production, which has been reported in many countries including the United States. One strategy to sustain sorghum yields under late-season water deficit is to exploit the stay-green trait. The phenotype of this trait is a persistence of green leaves for a longer period during grain filling. Thomas and Howarth (2000) proposed five mechanisms for the expression of stay green, one of which was a result of a delay in the initiation of leaf senescence after which the senescence process continues essentially in a normal manner. This appears to be the mechanism operating in sorghum. Further, those genotypes expressing stay green appear to do so only under water-deficit conditions. Vadez et al. (2011) and Jordan et al. (2012) concluded that the stay-green trait may result from Published in Crop Sci. 53: (2013). doi: /cropsci Crop Science Society of America 5585 Guilford Rd., Madison, WI USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher. crop science, vol. 53, september october

2 decreased water use early in the season, allowing water to be conserved to sustain a longer period of grain filling, hence the stay-green phenotype. One suggestion was to have decreased canopy size through decreased tillering so that early season water use is limited (Borrell et al., 2000). There are two alternate approaches for early-season water conservation that have not yet been considered as bases for the stay-green trait. Both approaches impose restrictions on transpiration rate, allowing possible water conservation for use later in the growing season. One approach results from an enhanced sensitivity to soil drying and the other from a limitation on transpiration rate under high vapor pressure deficit (VPD) conditions. The first approach is based on the observation that plants generally express a consistent pattern of transpiration rate in response to soil drying. Ritchie (1973) showed that initially plants growing on drying soil have no decrease in transpiration until the soil reaches a threshold of extractable water. Commonly, the threshold is in the range of 0.25 to 0.40 extractable soil water (Sadras and Milroy, 1996). Gholipoor et al. (2012a) examined the possible variability of the threshold among sorghum genotypes. In their comparison of 17 genotypes, they found that the threshold, expressed as a fraction of transpirable soil water (FTSW), varied from 0.32 to In related studies, Kholova et al. (2010) found, in a comparison of eight pearl millet (Pennisetum glaucum L. R. Br.) genotypes, that the threshold ranged from 0.30 to 0.47, and Gholipoor et al. (2012b) found, in a comparison of 36 maize (Zea mays L.) hybrids, a threshold range of 0.33 to Those genotypes with a high threshold seemingly have the capacity to conserve soil water during soil-drying events early in the growing season so that soil water remains during grain filling to allow sustained growth and expression of the stay-green phenotype. The second approach to water conservation is to have the transpiration rate of plants limited to a maximum rate, which would be expressed under high atmospheric VPD conditions. Therefore, under high VPD conditions, which can develop during the midday, water would be conserved for use later in the season and potentially allow expression of the stay-green trait. While many genotypes within species do not express a maximum transpiration trait and have continually increasing transpiration rates with increasing VPD, genotypes have been readily identified in sorghum that express this trait. Gholipoor et al. (2010) found that 17 of 26 sorghum genotypes they studied exhibited limited transpiration under high VPD. Within the 17 sorghum genotypes expressing the limited transpiration trait, the VPD at which the limitation was initiated varied from 1.6 to 2.7 kpa. Similar results were demonstrated in pearl millet, in that a genotype tolerant to drought expressed limited-transpiration trait at high VPD (>2.0 kpa) (Kholova et al., 2010). Gholipoor et al. (2012b) examined the response to increasing VPD of 35 maize hybrids and found 11 expressed the maximum transpiration trait with the breakpoint for the limitation on transpiration occurring at 1.7 to 2.5 kpa. Sinclair et al. (2005) simulated the potential benefit of the maximum-transpiration trait for sorghum over 100 yr at four locations in Australia. The simulation results showed yield increases when the trait was at yield levels less than 4.5 t ha -1 and only small decreases in yield above this level. These simulation results are similar to the observed response reported by Jordan et al. (2012) for genotypes expressing the stay-green trait. They found yield increases at yield levels below 6 t ha -1 and mixed results above this yield level. The objective of this study was to determine if the stay-green trait observed for sorghum genotypes is related to modified transpiration rates either when soil is drying or under high VPD conditions. Twelve genotypes were selected for this study on the basis of phenotyping under field observations. Seven of the genotypes expressed the stay-green phenotype and five expressed no stay-green response. These 12 genotypes were subject to a drydown test under controlled conditions to determine their soil water content at the threshold for the decrease in transpiration rate. Similarly, the 12 genotypes were tested under controlled conditions to determine their transpiration response to increasing VPD. Materials and Methods Stay-Green Identification Twelve sorghum genotypes (Table 1) were observed for expression of stay green and other physiological traits in field experiments in 2006, 2007, or 2010 at Manhattan, Kansas. Genotypes were sown in single rows spaced at 0.75 m. The experimental design was two randomized complete blocks. Three randomly selected plants for each genotype were tagged in each replication after panicle emergence for data collection. Chlorophyll content was measured using a SPAD meter (Minolta Model 502, Spectrum Technologies) on the middle of the leaf lamina of the third fully expanded leaf from the top at about 21 d after flowering. During the same time, maximum potential quantum efficiency Fv/Fm was measured using a pulse-modulated chlorophyll fluorometer (OS30, Opti- Science). Both sets of observations were made throughout the growing season. Here results are reported for each genotype at the same phenological stage of 21 d after flowering. Visual stay-green ratings on the basis of complete rows were taken during seed-filling period (about 21 to 25 d after flowering) and close to physiological maturity. A visual rating scale of 1 to 5 on the basis of degree of leaf yellowness and greenness was used, with a rating of 1 indicating 0 to 10% of yellow leaves, 2 indicating 20 to 40%, 3 indicating 40 to 60%, 4 indicating 60 to 80%, and 5 indicating 80 to 100%. Data from field experiments were analyzed using PROCMIXED procedures in SAS 9.3 (SAS) crop science, vol. 53, september october 2013

3 Table 1. Expression of stay-green and related traits of various genotypes from field experiments conducted in Manhattan, Kansas. Those values followed by different letters in a column were significantly different (P < 0.05). Chlorophyll content Photochemical efficiency Stay-green rating during seed filling Stay-green rating at physiological maturity Genotype SPAD units Fv/Fm 1 5 scale 1 5 scale Stay Green SC b a 1.57 de 2.33 bcd B f ab 1.41 de 2.49 bcd PI c a 1.24 de 1.99 d SC a ab 1.07 e 2.16 cd BQL a ab 1.41 de 2.86 bc Tx436/07MN d bc 1.74 d 2.99 b R bc a 1.57 de 2.99 b Non Stay Green Tx b cd 2.91 c 4.83 a Tx g d 3.24 bc 4.49 a Tx g e 3.41 bc 4.66 a Tx e e 3.74 ab 4.99 a Tx f de 4.07 a 4.83 a LSD Transpiration Response to Soil Drying Sorghum plants for the soil-drying experiment were grown in a greenhouse (Phytotron, North Carolina State University, Raleigh) with temperature regulated for a night temperature of 26 C and day temperature of 32 C. The plants were grown in 2.2-L plastic pots filled with soil to within 2 cm of the top of the pots. The soil was a sandy loam (11% clay, 34% silt, 55% sand). Analysis of the soil showed that it contained 0.35 g g -1 organic matter, 24 mg g -1 N, 0.18 mg g -1 K, and 5 µg g -1 P. Because of limited greenhouse space, the genotypes were randomly split into two sets for dry-down experiments. The two sets of experiments were sown on 3 Feb (Set 1) and 22 Feb (Set 2). There were eight pots for each genotype. Measurements of the temperature in the greenhouse gave the average day temperature as 34.2 C and average night temperature as 22.0 C. The pots were maintained in a well-watered condition. Sixteen days after sowing, each pot was thinned to one plant. The dry-down experiment was performed using the protocol originally described by Sinclair and Ludlow (1986). Ten days before the start of a dry-down experiment the plants were moved to a growth chamber with a maximum temperature of 34.2 C and a minimum temperature of 22.0 C, with a 14-h photoperiod. On Day 27 after sowing, when all genotypes had about four fully expanded leaves, all pots were overwatered and allowed to drain overnight. The following day each pot was enclosed in a white plastic bag with the bag opening bunched around the base of the stem and secured with a twist tie. An 8-mm-diameter 80-mm-long tube was inserted between the base of the plant and the plastic bag to facilitate watering of the pot during the experiment. Each pot was weighed after bagging and the weight was recorded as the initial pot weight. Thereafter, pots were weighed every day between 1400 and 1500 h. Daily transpiration was calculated as the difference in weight of each pot on successive days (Devi et al., 2010). Three of the eight pots of each genotype were selected to be well-watered control plants. One plant to be well watered was selected from the group of highest, middle, and lowest rank of plant transpiration rate within each genotype. The well-watered plants of each genotype were maintained at 100 g below the initial pot weight by replacing daily the amount of water lost from the pot to the 100-g deficit level. The soil in the five remaining pots was allowed to dry. To prevent rapid dehydration of the soil and to extend the drying cycle in these pots, water was added if necessary to a drying pot to keep the maximum daily net water decrease to 50 g. The transpiration of all pots on each day was calculated as the difference in water loss between successive days. To account for variable transpiration rates among plants within a genotype, a transpiration ratio for each pot on each day (TR i ) was calculated by dividing the transpiration for each drying pot (Tdry i ) by the average transpiration rate of the three well-watered pots Tww i. The subscript notes the day of measurement. Therefore, for each drying pot on each day the following was calculated. TR i = Tdry i /Tww i [1] To allow ready comparison among genotypes, the transpiration ratios were normalized for each pot so that their normalized transpiration ratios (NTR i ) were centered on 1.0 when the drying pot still was in the well-watered range. This normalization was done by calculating for each pot the average transpiration ratio for the first 3 or 4 d of the experiment (TRave). All daily transpiration ratios were divided by the initial average transpiration ratio to obtain for each day NTR i (Devi et al., 2010). NTR i = TR i /TRave [2] The dry-down experiment continued until the NTR of a pot decreased to 0.1. The water content of each drying pot was based on the total transpirable soil water, which was the difference in weight between the initial and final weight of the pot. The FTSW was calculated as the difference between daily weight and final weight, divided by the total transpirable water. Data were analyzed by doing a two-segment linear regression of NTR vs. FTSW combined for all the data of a genotype. This regression analysis was done using the nonlinear regression procedure (Ray and Sinclair 1997) in GraphPad Prism (GraphPad Software Inc.). An output of the regression analysis was the FTSW crop science, vol. 53, september october

4 threshold between the two linear segments representing the initiation of the decline in transpiration rates. Transpiration Response to Vapor Pressure Deficit Measurement of response to vapor pressure was done using the protocol presented by Gholipoor et al. (2010) in their comparison of sorghum genotypes. Plants were grown in pots constructed for attachment of VPD chambers in which transpiration measurements in response to different VPD levels were made. Plants were grown in pots constructed from 0.1-m-diameter and 0.32-m-long sections of polyvinyl chloride pipe (PVC). Pots were filled with 1.8 to 2 kg of compost garden soil (Miracle-Gro Lawn Products Inc.) containing slow-release fertilizer. The pots were placed in a greenhouse (North Carolina State University Phytotron, Raleigh, NC) regulated to temperatures of 30 C during the day and 26 C during the night. The plants were maintained under well-watered conditions. Once approximately four leaves had fully expanded on the plants, transpiration rate was measured under a range of VPD. Twelve VPD chambers were available for making the measurements, so in most cases four genotypes with three replications were measured at one time. One set of plants was measured for four genotypes and two sets of plants were measured for eight genotypes. The three pots of each genotype to be tested at one time were transferred from the greenhouse to a walk-in growth chamber 3 to 5 d before the transpiration measurements were to be done. The temperature in the growth chamber was held constant at 32 C during the experiment. The photosynthetic photon flux density in the growth chamber was about 650 μmol m -2 s -1. The soil surface was covered with aluminum foil to minimize soil evaporation during the measurements of plant water loss. A transparent, 21-L container was placed over each of the plants and loosely bolted to the toilet flange on the pot. The attachment of the chamber was not sealed because air was allowed to escape after flowing through the VPD chamber. Each chamber was fitted with a 76-mm-diameter computer box fan (Northern Tool and Equipment) to stir the air inside the chamber continuously. Also, a temperature and humidity sensor (Lascar Electronics) was mounted through the wall of each chamber to measure the air temperature and humidity in the chamber at 1-min intervals. Transpiration responses were measured on two consecutive days at three humidity levels on each day at three ranges of VPD: low ( kpa), medium ( kpa), and high ( kpa). The lowest VPD range was achieved by flowing air through the VPD chamber at a low rate (<0.8 L min -1 ), allowing water transpired from the plant to raise the humidity in the chamber. The medium VPD was achieved by increasing the airflow through the chambers to about 0.8 to 1.2 L min -1. The highest VPD treatment was achieved by first flowing air through a PVC column filled with silica gel to remove water vapor from the air and then flowing this dried air through the chamber in the range of 1.2 to 2.2 L min -1. The VPD treatments were applied sequentially, from the lowest to the highest, to avoid any lingering consequences of first exposing the plants to the high VPD treatment. The VPD in the chambers was allowed to stabilize for 30 min after introducing each humidity treatment. Following this stabilization period, the initial weight of the pot was measured on a balance with a resolution of 0.1 g (Model s1-8001, Denver Instrument). Then the plants were exposed to the humidity treatment for 90 min and reweighed. The differences in initial and final weights, and the duration of exposure to the VPD treatment, were used to calculate transpiration rate. The VPD was averaged during the treatment period on the basis of the measurements of temperature and relative humidity in the chamber. After completing measurements on the second day, the plants were dissected and the total area of the leaves on each plant was measured using a leaf area meter (LI-1300, Licor). Transpiration rate was expressed on a plant leaf area basis. All data were combined for a two-segment linear regression analysis (Prism 2.01, GraphPad, Software Inc.). If a difference in slope (p < 0.05) was not obtained in the regression, then all the data for that genotype were represented by a single linear regression. For those genotypes found to be represented by the two segments, the regression analysis generated the VPD breakpoint between the two linear segments. Results Stay-Green Identification Genotypes varied in the stay-green phenotype on the basis of chlorophyll content, chlorophyll fluorescence, and visual stay-green ratings (Table 1). For all three different traits, the genotypes could be clearly categorized into two distinct groups. Seven genotypes were identified expressing staygreen traits of higher SPAD values, higher Fv/Fm ratio, and higher stay-green rating (SC599, B35, PI , SC35, BQL41, Tx436/07MN, and R-45). Non-stay-green genotypes had lower SPAD, lower Fv/Fm ratio, and lower stay-green ratings (Tx623, Tx7078, Tx3042, Tx436, and Tx7000). Neither the range in grain yield nor mean yield differed between genotypes expressing stay green and those that did not express the trait (data not shown). Transpiration Response to Soil Drying The results of the soil-drying experiment, including the FTSW threshold, are presented for each of the 12 genotypes in Table 2. The two-segment linear model described well the results for all genotypes (R 2 = 0.96 to 0.99). The range in thresholds within both the stay-green and non-stay-green groups of genotypes was 0.38 to Tx436 was tested in both dry-down experiments, and there was consistency in the results of the two experiments. There was no apparent difference in the distribution of the soil-drying threshold between the stay-green and non-stay-green genotypes. Transpiration Response to Vapor Pressure Deficit The results of the VPD experiment are presented in Table 3. Five of the stay-green genotypes and two of the non-stay-green genotypes demonstrated the twosegment linear response to increasing VPD. The R 2 for these seven genotypes ranged from 0.78 to The breakpoint between the two segments ranged from 1.2 to crop science, vol. 53, september october 2013

5 2.9 kpa. The most extreme breakpoints were 1.17 kpa by BQL41and 2.91 by SC35. Two of the stay-green and three of the non-staygreen genotypes were found to have a linear increase in transpiration rate with increasing VPD and exhibited no breakpoint. The R 2 of the five linear response genotypes ranged from 0.72 to Hence, there was no clear segregation between the stay-green and non-stay-green genotypes on the basis of the expression or lack of expression of a breakpoint in transpiration rate with increasing VPD. Discussion The stay-green phenotype has been observed in some genotypes of sorghum in water-limited rainfed conditions and is thought to be associated with higher yielding potential under these conditions. It has been suggested by some (Vadez et al., 2011; Jordan et al., 2012) that the stay-green trait could be a result of a water conservation mechanism early in the growing season that allowed water to be available to the crop during late-season drought for sustained growth. Two physiological mechanisms that result in water conservation are an early decrease in stomata conductance with soil drying and a limited transpiration rate under high VPD. The objective of this study was to determine if either or both of these mechanisms were associated with the stay-green phenotype. There was a clear distinction between stay-green and nonstay-green genotypes in the field observations of chlorophyll content, chlorophyll fluorescence, and visual stay-green rating. In the comparison of seven stay-green genotypes and five non-stay-green genotypes, there was no segregation on the basis of either trait (decreased stomatal conductance or limited transpiration rate). That is, the threshold for the Table 2. Results of dry-down experiment for 12 sorghum [Sorghum bicolor (L.) Moench] genotypes: initial transpiration rate during the initial well-watered stage, days to reach endpoint of dry down, and fraction transpirable soil water (FTSW) threshold and confidence interval in the dry down. The standard error of the mean is presented following the ± for some of the data. Note: Tx436 was tested in duplicate experiments. Endpoint FTSW Confidence Genotype Transpiration of dry down threshold interval R 2 g plant 1 d 1 days p < 0.05 Stay Green SC ± ± a B ± ± a PI ± ± a SC ± ± ab BQL ± ± ab Tx436/07MN 66.7 ± ± b R ± ± b Non Stay Green Tx ± ± a Tx ± ± a Tx ± ± a Tx ± ± ab Tx ± ± ab Tx ± ± b Gholipoor et al. (2012a). decrease in transpiration rate with soil drying showed the same range for the stay-green and non-stay-green genotypes. Further, genotypes expressing the breakpoint in transpiration rate with increasing VPD were found among both staygreen and non-stay-green genotypes. There was no apparent consistent difference in the response to VPD among the two groups of sorghum genotypes. Table 3. Results of vapor pressure deficit (VPD) experiment for 12 sorghum [Sorghum bicolor (L.) Moench] genotypes: leaf area ± standard deviation (cm 2 plant -1 ) and regression results (breakpoint and slopes of two segments [mg H 2 O m -2 s -1 kpa -1 and R 2 ]) for the seven genotypes that fit the two-segment model. The results of the linear regression for five parents that were found not to fit the two-segment regression model are also presented. Parents Date of experiment Leaf area Breakpoint Slope 1 SE Slope 2 SE R 2 cm 2 plant -1 kpa Stay Green BQL41 15 Dec ± PI Dec ± SC599 Gholipoor et al., ± B35 Gholipoor et al., ± SC35 30 Nov ± R Nov ±50.2 linear Tx436/ Dec ±35.6 linear Non Stay Green Tx Mar ± Tx Dec ± BTx Dec ±34.6 linear Tx Mar ±48.2 linear Tx Mar ±78.6 linear Gholipoor et al. (2010). crop science, vol. 53, september october

6 The results of this study indicate that the two water-conserving mechanisms were not associated with expression of the stay-green phenotype. One possibility is that neither of these mechanisms was sufficiently expressed in the field studies to have a major impact on the expression of stay green. For the field conditions in which these genotypes were phenotyped, it appears the expression of stay green involves a mechanism(s) other than the ones studied here. One possibility is that the water conservation aspects of the stay-green may be only minor contributors and there may be a mechanism directly involved in the nitrogen balance resulting in greater retention of nitrogen in the leaves (van Oosterom et al., 2010a,b). These results did confirm previous observations of a wide range of responses among sorghum genotypes in both the threshold for the decrease in transpiration rate with soil drying and the response to VPD. The threshold range for the decrease in transpiration of these 12 genotypes was within the distribution reported by Gholipoor et al. (2012a) of FTSW 0.32 to Three genotypes in the current study were found to have especially high thresholds (Tx7000, Tx436/07MN, R-45), which could make these candidate genetic resources for incorporating this water conservation mechanism. This study identified BQL41 as having an especially low breakpoint in the response to increasing VPD. This genotype was found to have a breakpoint of 1.17 kpa, which was well below the lowest threshold of 1.6 kpa reported by Gholipoor et al. (2010). However, the very low threshold of BQL41 in itself would likely only be a benefit in very dry environments because of the possibility of lost photosynthetic activity. Nevertheless, this genotype might be especially useful as a parental source for introducing the maximum transpiration trait into germplasm. Acknowledgments Authors acknowledge financial support from the Kansas Grain Sorghum Commission. Contribution no J from the Kansas Agricultural Experiment Station. References Borrell, A.K., G.L. Hammer, and A.C.L. Douglas Does maintaining green leaf area in sorghum improve yield under drought? I. Leaf growth and senescence. Crop Sci. 40: doi: /cropsci x Deb, U.K., and M.C.S. Bantilan Impacts of genetic improvement in sorghum. In: R.E. Evenson and D. Gollin, editors, Crop variety improvements and its effect on productivity The impact of international agricultural research. CAB International, Wallingford, Oxon, UK. p Devi, M.J., T.R. Sinclair, and V. Vadez Genotypic variability among peanut (Arachis hypogea L.) in sensitivity of nitrogen fixation to soil drying. Plant Soil 330: doi: / s Jordan, D.R., C.H. Hunt, A.W. Cruickshank, A.K. Borrell, and R.G. Henzell The relationship between stay-green trait and grain yield in elite sorghum hybrids grown in a range of environments. 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Funct. Plant Biol. 38: doi: /fp11073 van Oosterom, E.J., A.K. Borrell, S.C. Chapman, I.J. Broad, and G.L. Hammer. 2010a. Functional dynamics of the nitrogen balance of sorghum: I. N demand of vegetative plant parts. Field Crops Res. 115: doi: /j.fcr van Oosterom, E.J., S.C. Chapman, A.K. Borrell, I.J. Broad, and G.L. Hammer. 2010b. Functional dynamics of the nitrogen balance of sorghum: II. Grain filling period. Field Crops Res. 115: doi: /j.fcr crop science, vol. 53, september october 2013