A physiological Explanation of Drought Effect on Flag-Leaf Specific Weight and Chlorophyll Content of Barley

This study was carried out in Kalar technical institute, Sulaimani Polytechnic University in Garmian region, Iraq during 2016-17 and 2017-18 seasons. Five hybrid genotypes of barley were tested under drought and irrigated conditions to detect the flag-leaf specific weight, chlorophyll content index (SPAD; The Soil Plant Analysis Development chlorophyll meter) and the period from anthesis to physiological maturity. Across both years 2016-17 and 2017-18, there was no effect of drought on flag-leaf specific weight, however, SPAD was reduced by 4.77 and the period between anthesis and maturity was shortened by almost two days. Genotype 3//14 scored the highest values of flag-leaf specific weight (7.765 mg cm 2 )and SPAD (25.56), and stayed green for the longest period (28.69 days) (P=0.05), showing its ability to be more tolerant to moisture reductionas compared to the other tested genotypes. In order to explain the physiological mechanisms among the assessed traits under both irrigated and drought conditions, linear regression analysis was applied for both seasons and averaged over seasons. A positive linear relationship was shownbetween flag-leaf specific weight and SPAD under both irrigated (R 2 =0.83; P=0.03) and drought (R 2 =0.76; P=0.05) conditions, explaining the high flag-leaf chlorophyll content resulting from high specific weight of leaves. The high flag-leaf specific weight was also associated with longer periods for leaves to stay-green after anthesis, under both irrigated (R 2 =0.91; P=0.01) and drought (R 2 =0.79; P=0.04) conditions, which provides a great chance to accumulate more resources of carbohydrates and protein in the grain and, consequently,a higher throughput of yield.


Introduction
Barley (Hordeum vulgare L.) is one of the important cereals in many dry areas of the world and it is necessary for the livelihoods of many farmers [1]. It is one of the major cereal crops that is primarily grown for its grain and used for animal feed [2]. Water stress is one of the vital limiting factors in crop production worldwide. In breeding programs, in order to enhance the drought resistance of a crop plant, it is necessary to have knowledge related to the physiology of drought tolerance mechanisms [3]. Drought is considered as one of the most effective abiotic stresses limiting agricultural production worldwide. Drought stress during the grain-filling period decreased the flag leaves' net photosynthetic rate of barley [4]. The sensitivity of drought effect 'was reported to occurjust before spike emergence stage' [5], particularly in environments where drought is encountered at the end of the plant'slife cycle [6]. Flag leaf is a primary source of carbohydrate production for grain filling and yield due to its short distance to the spike and the fact that it stays green for longer times than the rest of the leaves [7]. A previous study [8] found that some flag leaf traits, such as lengths and width, were inherited quantitatively. Understanding the role of physiological and morphological traits of flag leaf on yield will provide a new insight in crop growth and development [9].
Photosynthesis is the main source of grain yield and dry matter production in crop plants. It is also an essential process to maintain crop growth and development. Photosynthetic systems in higher plants are most sensitive to drought stress [10]. It was reported that measuring photosynthetic traits such as chlorophyll content might estimate the influence of environmental stress on crop growth and yield [11,12]. The objective of the present experiments was to study the effects of drought on flag leaf area and chlorophyll content in barley, and to physiologically explain the mechanism of their relationships under drought-prone environment.

Materials and Methods Plant materials and environmental conditions
Two experiments were carried out for two seasons of 2016-17 (Feb 2017 -May 2017; referred to hereafter as 2017) and 2017-18 (Dec 2017 -May 2018; referred to hereafter as 2018) at Kalar technical institute (at longitude line 45º 22′ 681″ east, latitude line 34º 21′ 558″ north, and elevation level of 178 meters). Five introduced varieties were obtained from Kalar Agricultural Research Station, which were originally developedwith different sensitivities for drought conditions by the International Centre for Agricultural Research in the Dry Areas (ICARDA) in Syria. Five hybrids of F 2 two-rowed barley (Hordeum vulgare L.) were then obtained from crossing a local variety in Garmian region with those developed by ICARDA, using a previously investigated full diallel cross [13].The hybrids were, namely, Local//Zanbaka (3//18), Local//ARTa/3/Avar (3//14), Local//Roho/Zanbaka (3//5), Local//Avar/H/Sout (3//1) and Local//Tadmor/Roho (3//4). The study region was of a semiarid climate [14] with anAridisols soil (characteristic of arid regions, containing typically saline or alkaline soils with low level of organic matter). Temperature was hyperthermic [15] based on day time temperature, and average daily temperatures (maximum + minimum temperature divided by 2) during the seasons were in the range 12.4-28.5 o C in 2017 and 6.5-31.3 o C in 2018. The soil was slightly moist or aridic (Torric) which requires irrigation for agricultural use [16]. The total rainfall in the region was 226.1 mm in 2017 and 287.4 mm in 2018. Figure

Experimental design and statistical analysis
Randomised block, split-plot design was used, including two main-plots and twenty sub-plots (5 rows x 4 columns) with four replicates (blocks) in each main plot. Irrigation treatments (fully irrigated and unirrigated) were randomised on main-plots. Genotypes were randomised on sub-plots (1 m 2 ). GenStat 19th Edition [17] was used for statistical analysis of variance (ANOVA) by applying a splitplot design for both years and cross-year mean data. Linear regression analysis and graphs were carried out using the GraphPad Prism 8.0.0 software package to calculate the relationships between all variables among years and for the cross-year mean [18].

Traits measurement Number of days from anthesis to maturity dates (AD-MD; day)
Anthesis date (GS61; Mid-April in 2017 and Early-April in 2018) and maturity date (GS89; Mid-May in 2017 and Early-May in 2018) were measured based on the decimal code of growth stages (GS), as previously described [19]. Anthesis date was visually assessed for the whole plant in each subplot, and a growth stage was taken when more than 50 % of the main shoots were at the anthesis date. Physiological maturity was also assessed based on the date when green area of the stem was less than 25%. Number of days from anthesis to maturity date (AD-MD) was then calculated by counting the total days from the date of anthesis till maturity date for each genotype. Leaf chlorophyll content (SPAD), from GS61-14 days to GS61+14 days, was measured weekly on the main shoots for three plants in each plot for both years (2017-2018) using a chlorophyll content meter (CCM-200, OPTI-SCIENCES, Japan). The average chlorophyll content index was then used for data analysis. The readings were taken when the sky was clear and the leaves were well illuminated between 10.00h to 14.00h of daily hours [20].

Number of days from anthesis to maturity dates (AD-MD; day)
There was no significant drought effect on the duration between anthesis and maturity (P=0.10) in 2017 (Table-3). The duration for the genotypes ranged from 26.25 days for 3//5to 28.5 days for 3//14 under irrigated conditions, and from 24.25 days for 3//18 to 26.75 days for 3//14 and 3//1 under unirrigated conditions, with the differences being significant (P=0.05). The interaction between irrigation and genotype showed no significant duration differences (P=0.55; Table-3). Drought reduced the maturity date by 2.2 days (P=0.04) in 2018. There were no significant differences in the duration values between the genotypes under irrigated and unirrigated conditions (P=0.79; Table-3). Averaging over years, drought significantly reduced the period from anthesis to maturity from 28.68 to 26.90 days (P=0.005). Genotypes showed duration values that ranged from 27.75 days for 3//1 to 29.63 days for 3//14 and from 26.13 days for 3//18 to 27.75 days for 3//14 under irrigated and drought conditions, respectively, with the differences being significant (P=0.007). There were also significant differences between years (P=0.004), but not between genotypes (P=0.22).

Discussions
Environmental data in the studied region showed higher humidity condition in 2017 than in 2018 over the grain filling period (February to April). Averaging over seasons, leaf specific weight in both years was not affected by drought,which is expected when drought occurs in the late growing season when leaves are fully emerged [21]. However, relative chlorophyll content (SPAD) was significantly decreased in both years,which can be attributed to limited water availability after anthesis [22]. Genotype 3//14 had the highest value of SPAD in both seasons, whichmight be due to high flag-leaf specific weight which helps in increasing photosynthetic activity and higher grain yield. Drought shortened the cross year mean period between anthesis and maturity by almost two days, possibly through causing advanced physiological maturity [23]. Regression analysis revealed a positive association between flag-leaf specific weight and SPAD, which clarified the importance of leaf morphology and thickness in order to have a high rate of photosynthesis activity [24]. Averaging over seasons, the specific weight of flag-leaves was significantly correlated with the number of days between anthesis and physiological maturity under both irrigated and drought conditions. Although drought fastens leaf senescence and advances maturity, thegenotypes with higher flag-leaf specific weight had longer stay-green periods and were later senesced [25].

Conclusions
The physiological mechanisms behind the photosynthetic process under water stress play the main role for a better grain yield in barley. In this study, chlorophyll content index (SPAD) appeared to be positively associated with flag-leaf specific weight, indicating the importance of this trait in selecting superior genotypes inbreeding programs with respect to flag-leaf area. Flag-leaf senescence duration after anthesis was also extended by the effect of flag-leaf specific weight under both irrigated and drought conditions. For these reasons, flag-leaf specific weight can be recommended to be an indicator for the best yield under drought environments.