Spike developmental stages and ABA role in spikelet primordia abortion contribute to the final yield in barley (Hordeum vulgare L.)
Salinity is a significant environmental stress factor limiting crops productivity. Barley (Hordeum vulgare L.) has a natural tolerance to salinity stress, making it an interesting study object in stress biology research. In the present study, for the first time the effect of salinity stress on barley inflorescence developmental stages was investigated. Five spring barley genotypes irrigated with saline water (12.5 ds/m NaCl) were compared to controls treated with normal tap water. We measured abscisic acid (ABA) concentrations in the apical, central and basal sections of the immature inflorescence at green anther (GA) stage. The role of ABA in spikelet primordia development, atrophy and abortion and final yield was evaluated.
A time course experiment starting from double ridge until green anther (GA) stages revealed that salinity reduced the length of spike developmental stages in all genotypes causing shortened of the plant life cycle. The shortened plant life cycle negatively affected plant height and number of tillers/plant. Salinity also affected spikelet primordia development. In both control and salinity treated plants apical spikelet abortion started in late awn primordium (AP) stage. However, under salinity treatment, significantly more spikelets were aborted, thus directly affecting plant yield potential. ABA, which plays a role in the spikelet/floret abortion process, was markedly elevated in the base and apex of salt treated spikes correlating with an increased spikelet abortion in these regions.
Overall, salinity treatment reduced all plant and yield-related parameters investigated and turned some of the correlations among them from positive to negative or vice versa. Investigations of ABA role in floral development and phase duration of barley spike showed that, ABA regulates the spikelet/floret abortion process affecting the yield potential under salinity and control conditions.
KeywordsBarley Salinity ABA Spike development Primordia abortion Spikelet/floret abortion
scanning electron microscopy
days after planting
Abiotic stresses due to salinity occurs naturally (Dai 2011) but has become a growing global problem due to human activities such as salt mining (Ghassemi et al. 1995) and poor irrigation systems (Marcum and Pessarakli 2006). At present more than 800 million hectares of agricultural land are affected by salinity and/or sodicity stress (Munns 2005; Farooq et al. 2015). In arid and semiarid countries, agricultural production is limited by water availability and the resources of water are insufficient for the growing human population. As fresh water is allocated in priority for drinking purposes, irrigation water is often of poor quality. Nowadays, it has been estimated that about 20% of the cultivated land worldwide is affected by salinity (Jamil et al. 2011). The corresponding proportion of irrigated agricultural land is 33%, expected to reach 50% of the arable land by the year 2050 (Jamil et al. 2011). The growing salinity problem in arid and semi-arid regions needs an urgent solution where research aiming to understand the effects of salinity on cereals production should be combined with genetic efforts to develop salt tolerant crops for the future of agriculture (Shannon 1984; Owens 2001; Kausar et al. 2013). Biochemical pathways, morphological and physiological processes including seed germination, growth and development are affected by salinity (Willenborg et al. 2004) causing yield and quality reduction (Basalah 2010; Bagues et al. 2018). However, plant species differ in their response to salinity stress (Torech and Thompson 1993; Sarabi et al. 2017).
At the awn primordium stage six-rowed barley displays more spikelets/florets primordia per spike than two-rowed barley (Whingwiri and Stern 1982; Kirby and Appleyard 1987; Kernich et al. 1997; Miralles et al. 2000; del Moral et al. 2002; Arisnabarreta and Miralles 2006). Inflorescence development and growth, phase duration and organ patterning are influenced by phytohormones such as auxin (IAA), cytokinin (CK) and abscisic acid (ABA) (Su et al. 2011; Matsoukas 2014; Youssef et al. 2017). ABA also is the primary hormone that mediates plant responses to stress such as drought and salinity (Wu et al. 1997; Wilkinson and Davies 2010; Lee and Luan 2012). It has been suggested that the ABA level correlates with the plant resistance to stress (Maslenkova et al. 1993; Lee and Luan 2012) including salinity stress (Nilsen and Orcutt 1996; Suzuki et al. 2016). ABA is supposedly involved in the induction of the synthesis of the 26-kDa protein Osmotin which accumulation depends on the presence of NaCl (Bressan et al. 1985). ABA may also influence stomatal conductivity affecting tissue hydraulics (Collins and Kerrigan 1974; Davies and Zhang 1991; Freundl et al. 2000; Hose et al. 2002; Du et al. 2013) growth in response to drought or salinity through changes in cell wall extensibility (Bacon 1999; Cramer et al. 1998; Dodd and Davies 1996; Thompson et al. 1997) and apoplastic pH in plants (Bacon et al. 1998). Other studies have focused on the effect of ABA accumulation and a decrease in IAA and CK on the progression of senescence in salinized plant organs (Albacete et al. 2008; Ghanem et al. 2008). Salinity affects both vegetative and reproductive developmental stages and reduces shoot growth and the number of florets per ear. Furthermore, salinity increases sterility and changes the time of flowering and maturity in grasses (Läuchli and Epstein 1990). In the present work we studied the response of inflorescence developmental stages, spikelet primordia development, and ABA concentrations in the inflorescence of five spring barley genotypes to salinity stress and related this to final yield.
Materials and methods
Year of release
Local Tunisian variety
Adapted to the driest regions
Selected by INRAT in 2011
Resistance to fungal diseases
Selected by INRAT in 2009
Used as fodder
Selected by INRAT in 1996
Suitable for wetlands
Introduced from ICARDA in 1982
Adapted to semi-arid regions
Growing conditions and salinity treatment
The five barley genotypes were grown in IPK-Gatersleben greenhouse under long day conditions 16 h/8 h (day/night) and temperature of ~ 20 ± 2 °C during the day and ~ 16 ± 2 °C during the night. Per genotype two sets of 96 seeds were germinated in 96 wells plates, one watered with tap water as control and the second treated with saline water (12.5 ds/m NaCl, selected based on preliminary where the plants died at more than 12.5 ds/m NaCl concentration) starting from the day of planting. When seedlings reached three leaves stage, they were transferred into 14 cm diameter pots, irrigated with either tap water or saline water. Agricultural practices were performed as recommended, including pest, disease and weed control.
Plant and spike phenotyping
To establish spike developmental stage, every second day after transfer into 14 cm pots, two plants of each genotype were dissected under a stereomicroscope (Stemi 2000-c, Carl Zeiss Micro Imaging GmbH, Gottingen, Germany). To compare spike developmental under salinity stress with control conditions, spikes of five or more plants at the developmental stages: Double Ridge (DR), Triple Mound (TM), Glum Primordia (GP), Stamen Primordium (SP), Lemma Primordium, Awn Primordium (AP), White Anthers (WA) and Green Anthers (GA) according to Kirby and Appleyard (1987), were collected for electron micrographs. After anthesis ten control and ten salinity treated plants were scored for plant height, number of tillers and main spike length. After complete maturity all spikes from each plant were collected for yield. Yield-related parameters seed length, seed width, seed area, number of seeds/plant, plant seeds weight and 1000-Grain weight) were measured. The experiment was repeated three times in the same green house under the same conditions.
Scanning electron microscopy (SEM)
For SEM analysis, isolated barley spikes were fixed with 4% formaldehyde in 50 mM phosphate buffer, pH 7.0 for 16 h. After dehydration in a graded ethanol series and critical point drying in a Bal-Tec critical point dryer (Bal-Tec AG, Balzers, Switzerland), spikes were gold sputtered in an Edwards S150B sputter coater (Edwards High Vacuum Inc., Crowley, West Sussex, UK) and examined in a Hitachi S-4100 SEM (Hisco Europe, Ratingen, Germany) at 5 kV acceleration voltage. Digital recordings were made and stored as Tiff-image files.
To compare ABA concentration along the developing spike, of each genotype 12 to 16 spikes at GA stages were collected and sectioned into basal, central, and apical parts (Youssef et al. 2017). After freeze-drying 20 to 50 mg dry weight was used to extract ABA according to Kojima et al. (2009) and Seo et al. (2011). ABA analysis by GC/MS (Shimadzu GC 2010 A chromatograph) was performed as described in Okamoto et al. (2009). Data from four biological replicates were analyzed and significance values were calculated.
The experiment was arranged as a completely randomized design with ten replicates per genotype per treatment (control and salinity). Main effects of genotypes, control and salinity treatments, along with the corresponding interactions were tested using two-way analysis of variance (ANOVA). Significance of differences between means was estimated with Tukey’s HSD (Honest Significant Difference test). Pearson correlation coefficients for pairwise comparisons between all traits were computed. All statistical analyses in this study were conducted using R 3.5.3 (R Core Team 2018).
Effect of salinity treatment on plant height and number of tillers
Salinity affects the length of spike developmental stages
Salinity reduces the yield potential
Salinity affects ABA concentration in the immature spike
Effect of salinity treatment on yield traits
Under control conditions, data show different correlations between studied traits (Fig. 6a). Plant height was highly positive correlated with main spike length, seed area and seed length. Similarly, positive correlation was found for seed weight/plant which was highly correlated with TSW, seed width and number of seeds/plant. TSW was greatly correlated with seed width, the same for seed area and seed length, both correlations were positive. However, number of tillers/plant and seed length were negatively correlated with TSW and seed width. Under salinity stress treatment, new correlations between the traits comparing to control were detected (Fig. 6b). On the contrary of control conditions, plant height was positive correlated with number of seeds/plant and seed weight/plant, however, this positive correlation reduced with seed area and seed length. For main spike length, a negative correlation with number of seeds/plant and seeds weight/plant under control conditions turned into a positive correlation under salinity stress, a positive correlation with TSW was established. Number of seeds/plant became negatively correlated with TSW and seed width, the positive correlation between number of seeds/plant and seed weight/plant was more important under salt stress conditions than control. Regarding to seed area, two new positive correlations were generated under stress comparing to control.
The study for the first time describes the effect of salinity stress (EC level 12.5 ds/m) on spike developmental stages, spikelet primordia development and spike ABA concentrations in relation to yield in spring barley. The salinity caused reduction in the plant growth may be due to the negative effect of salt on many metabolic processes including protein nucleic acid and polyamine synthesis, transpiration, stomatal conductance, photosynthesis (Mittal and Dubey 1991; Reggiani et al. 1994; Netondo et al. 2004; Abbas et al. 2015; Bagues et al. 2018). Certain ions can restrict the absorption of water by plant roots (Mansour 1994; Ahmed et al. 2013), and/or induce an imbalance in phytohormone levels either through altered biosynthesis or a change in turn-over rates (Amzallage et al. 1992; Dunlap and Binzel 1996). Decreased plant height under the salinity might be due to the accumulation of salts in the cell wall limiting cell wall elasticity and cell elongation (Naseer et al. 2001; Taghipour and Salehi 2008; Colla et al. 2012) and resulting in stunted shoots (Aslam et al. 1993). Salinity stress also caused a significant reduction in number of tillers in all tested barley genotypes. These results concurred with the findings of Nicolas et al. (1994), Zhao et al. (2007), Shahzad et al. (2012) and Bagues et al. (2018).
In our previous study (Youssef et al. 2017) we described the hormonal role in regulating floral organ patterning and phase duration during barley inflorescence and shoot development. Spike development is mainly influenced by phytohormones such as IAA, CK and gibberellins (Pearce et al. 2013). Atrophy and degradation of spikelet primordia in the apical and basal sections of the spike during green anther stage are additional proof of a hormonal role in spike and spikelet/floret development. The high concentrations of ABA in these sections at GA stage cause a local inhibition of floret development (Wang et al. 2000). In the central part of the spike, however, the lower concentration of ABA found in this study and higher concentration of gibberellins (our previous study, Youssef et al. 2017) allows the development of fertile flowers and grain setting (Fig. 7). Similar results were also reported by Wang et al. (1999), Cao et al. (2000), Youssef et al. (2017) and Youssef and Hansson (2019). This ABA dependent modification of the grain number at the apical and basal parts of the spike could be the mechanism by which the barley spike adapts its yield potential. Atkinson et al. (2013) reported on a negative correlation between the susceptibility to abiotic stress (salinity and drought) and ABA concentrations. In accordance to this we found highest ABA concentrations in the saline sensitive genotype Lemsi and lowest concentrations in the saline insensitive genotype Rihane (Fig. 5). In addition to the ABA effect, the spikelets/florets atrophy could be due to salt stress initially inducing osmotic stress and causing reduced water availability for the spikelets primordia, followed by ion toxicity due to nutrient imbalances in the cytosol causing spikelets/florets degradations and abortions.
In the present study, we also showed that salinity treatment adversely affects number of seeds/plant, weights of seeds/plant and TSW compared with the respective values in non-stressed control plants (Additional file 3: Table S2). The significant reduction in the grain yield under salt stress may be due to (i) an inhibition of tillering capacity (Fig. 2) causing a reduction in the number of spikes/plant as previously concluded by Al-Khafaf et al. (1990), Sakr et al. (2007) and Boussen et al. (2016), and (ii) enhanced degradation of the spikelet primordia through increasing of ABA concentrations in the apical and basal parts of the spikes (Figs. 4, 5, 7) reducing the number of seeds/spike. The detrimental and injurious effects of salinity on the growth productivity of barley shown here are in accordance with previous reports. Kumar et al. (1987) and Hank et al. (1989) showed that increasing salinity level in the irrigation water decreased growth and yield components of the plant. Holloway and Alston (1992) reported that salt stress decreased tillering, dry matter production and grain yield while Zeng and Shannon (2000) on rice and Sakr and El-Metwally (2009) on wheat, revealed that the reduction of tiller number/plant and spikelet number/panicle were the major causes of yield loss under salinity stress conditions. Sakr et al. (2004) suggested that the reduction in grain yield is largely due to a decrease in grain set which may be attributed to a decrease in the viability of pollen or in the receptivity of the stigmatic surface or both. Grattan et al. (2002) showed that salinity had negative impacts on number of spikes, tillers and spikelets per plant, floret sterility, individual grain size, and heading. We hypothesize that the reduction in grain yield of barley under salinity stress may be attributed to a diminished cell division and cell expansion in the spike, caused by altered concentrations of certain plant hormones like ABA. The ultimate result is a shortening of the spike developmental stages and a decreased production of developed spikelets and subsequent pollen grains. Hormones cross talking and their biological and genetic effects on spike and spikelet development appears a promising field for future work.
Overall, salinity treatment reduced all yield-related parameters investigated and turned some of the correlations among them from positive to negative or vice versa. Investigations of ABA role in floral development and phase duration of barley spike showed that, ABA regulates the spikelet/floret abortion process affecting the yield potential under salinity and control conditions.
We thank the Aridlands and Oases Cropping Laboratory of Medenine, Tunisia, and IPK Genebank, Gatersleben, Germany for providing germ plasm for the study. We thank T. Schnurbusch for providing Stemi 2000-c, Carl Zeiss Micro Imaging GmbH, Gottingen, Germany for doing spike developmental part of the work in his group. We also would like to thank the anonymous reviewers for their constructive and supportive evaluations while improving this manuscript. This work was supported by grants from the Tunisian national Ministry of Higher Education and Scientific Research and the University of Carthage to F.B.
AB and HMY conceived and designed the research. FB, MA, FG, AF, TR and HMY performed experiments. FB, MA, HMY and MH analyzed data. FB, MA, HMY, MH and AB wrote the manuscript with contributions from all coauthors. All authors read and approved the final manuscript.
This work was supported by grants from the Tunisian national Ministry of Higher Education and Scientific Research and the University of Carthage to F.B.
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Authors agree to the terms of the Springer Open Copyright and License Agreement.
The authors declare that they have no competing interests.
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