3.3 Summary
4.2.5 Comparison between Morphological and Physiological Traits
Following the analyses of both morphological and physiological traits, significant variation between B. oleracea genotypes in response to salt stress was observed. An effort has been made to compare the two measurements with the view to unravel possible relationships
Geno ID Species 8wk24hr pt_K:Na 8wk24hr pt_Ca:Na 10wk_K:Na 10wk_Ca:Na Response C10001 B. ole(DHSL150) 1.083 0.393 0.604 0.659 Susceptible
C13013-DH 1.988 0.596 2.895 2.187 Tolerant
C10025-DH 1.595 0.549 3.014 2.294 Tolerant
C10121-DH 1.636 0.722 1.759 0.269 Mildly Tolerant
C13001-DH 2.819 0.811 1.871 1.531 Mildly Susceptible
C07079A B. oleracea-S1 11.119 3.853 8.728 6.676 Tolerant
C07060 B. oleracea-S1 3.450 0.802 12.061 5.784 Tolerant
C07007 B. bourgaei-S1 1.122 0.410 1.516 1.009 Mildly Tolerant
based on the observed variation. Therefore, a Pearson correlation analysis was conducted between the two traits in all the individual B. oleracea lines. The strength of the correlation (R2) will be used to compare with between and the observed phenotype and to speculate
possible mechanistic relations. Parents line and their derived DH lines will also be compared so as to derive any shared physiological mechanism between the parents and DH lines respectively.
From the analysis, the cultivated rapid cycling DHLS150 line was shown to be weakly correlated in plant height, leaf fresh weight and strong in dry weight in relation to Na+, K+
(R2 = 0.273, 0.639 & 0.937), and negatively corelated with respect to leaf area (Table 4.4).
We observed an increase in both leaf fresh and dry weights in DHSL150. In contrast, leaf area correlates negatively (R2 = -0.0761) and that could be as a result of high salt (Na+)
absorbed as it affects cell expansion and stomatal closure. In one of the DH line; C13013- DH derived from the DHSL150, has shown positive correlation in both plant height and fresh weight with respect to Na+ level (R2 = 0.318 & 0.784) and had also correlated
negatively to K+ level. This could explain the differences observed between the parent line
Table 4.4: A Pearson correlation between morphological and physiological traits in the cultivated DHSL150 and DH line C13013-DH
Key: Morphological Traits: PH = Plant Height, LFW = Leaf Fresh Weight, LDW = Leaf Dry weight, LA = Leaf Area. Physiological Traits: 24hr_Na = Na+ 24hr post 1st
salt treatment, 24hr_K = K+ 24hr post 1st salt treatment, 24hr_Ca = Ca2+ 24hr post 1st salt treatment, 8wk_Na = Na+ 2 weeks post 1st salt treatment, 8wk_K = K+ 2 weeks
post 1st salt treatment, 8wk_Ca = Ca2+ 2 weeks post 1st salt treatment, 8wk24hr_Na = Na+ 24hr post 2nd salt treatment, 8wk24hr_K = K+ 24hr post 2nd salt treatment,
8wk24hr_Ca = Ca2+ 24hr post 2nd salt treatment, 10wk_Na+ = 2 weeks post 2nd salt treatment, 10wk_K = K+ 2 weeks post 2nd salt treatment, 10wk_Ca = Ca2+ 2 weeks post
Similarly, the wild S1 line; B. bourgaei-S1 (C07007) has shown a positive correlation between plant height, leaf fresh and Na+, K+, and Ca2+ (Table 4.5) but correlates negatively
in leaf dry weight and leaf area. This is in contrast to what was observed in the DHSL150, which shows a positive relation in Na+ level and reduction in plant height. The wild S1
C07007 has shown an improved growth against its control line. This may further suggest clear differences in both physiology and morphological mechanisms employed by the parents' lines to handle excess Na+ and at the same time improved growth. One of their
derived DH line; C10025-DH that shows susceptibility in growth has shown a negative correlation between Na+ and plant height and had reduced leaf dry weight and leaf area,
both correlate positively with the level of Na+ and K+. This corroborates our argument that
the DH lines may show similar mechanisms in both morphological and physiology, however, differ to some certain extent due to possible allelic recombination and introgression.
Table 4.5: A Pearson correlation between morphological and physiological traits in the wild S1 B. bourgaei-S1 (C07007) and DH line C10025-DH
Key: Morphological Traits: PH = Plant Height, LFW = Leaf Fresh Weight, LDW = Leaf Dry weight, LA = Leaf Area. Physiological Traits: 24hr_Na = Na+ 24hr post 1st salt treatment, 24hr_K = K+ 24hr post 1st salt treatment, 24hr_Ca = Ca2+ 24hr post 1st salt treatment, 8wk_Na = Na+ 2 weeks post 1st salt treatment, 8wk_K = K+ 2 weeks post 1st salt treatment, 8wk_Ca = Ca2+ 2 weeks post 1st salt treatment, 8wk24hr_Na = Na+ 24hr post 2nd salt treatment, 8wk24hr_K = K+ 24hr post 2nd salt treatment, 8wk24hr_Ca = Ca2+ 24hr post 2nd salt treatment, 10wk_Na+ = 2 weeks post 2nd salt treatment, 10wk_K = K+ 2 weeks post 2nd salt treatment, 10wk_Ca = Ca2+ 2 weeks post 2nd salt treatment.
The wild S1 B. oleracea-S1 (C07060) also showed a reduction in plant height and leaf area that correlated negatively with Na+, K+, and Ca2+ (Table 4.7), This differ compared to its
DH line, C10128-DH which showed a positive correlation in both plant height and leaf dry weight. Both the parent line and DH line gave a negative correlation between leaf area which corroborates with morphological effects observed in the reduction of leaf area associated with excess salt.
Table 4.6: A Pearson correlation between morphological and physiological traits in the wild S1 B. oleracea (C07079A) and DH line C10121-DH
Key: Morphological Traits: PH = Plant Height, LFW = Leaf Fresh Weight, LDW = Leaf Dry weight, LA = Leaf Area. Physiological Traits: 24hr_Na = Na+ 24hr post 1st salt treatment, 24hr_K = K+ 24hr post 1st salt treatment, 24hr_Ca = Ca2+ 24hr post 1st salt treatment, 8wk_Na = Na+ 2 weeks post 1st salt treatment, 8wk_K = K+ 2 weeks post 1st salt treatment, 8wk_Ca = Ca2+ 2 weeks post 1st salt treatment, 8wk24hr_Na = Na+ 24hr post 2nd salt treatment, 8wk24hr_K = K+ 24hr post 2nd salt treatment, 8wk24hr_Ca = Ca2+ 24hr post 2nd salt treatment, 10wk_Na+ = 2 weeks post 2nd salt treatment, 10wk_K = K+ 2 weeks post 2nd salt treatment, 10wk_Ca = Ca2+ 2 weeks post 2nd salt treatment
Table 4.7: A Pearson correlation between morphological and physiological traits in the wild S1 B. oleracea-S1(C07060) and DH line C10128-DH
Key: Morphological Traits: PH = Plant Height, LFW = Leaf Fresh Weight, LDW = Leaf Dry weight, LA = Leaf Area. Physiological Traits: 24hr_Na = Na+ 24hr post 1st salt treatment, 24hr_K = K+ 24hr post 1st salt treatment, 24hr_Ca = Ca2+ 24hr post 1st salt treatment, 8wk_Na = Na+ 2 weeks post 1st salt treatment, 8wk_K = K+ 2 weeks post 1st salt treatment, 8wk_Ca = Ca2+ 2 weeks post 1st salt treatment, 8wk24hr_Na = Na+ 24hr post 2nd salt treatment, 8wk24hr_K = K+ 24hr post 2nd salt treatment, 8wk24hr_Ca = Ca2+ 24hr post 2nd salt treatment, 10wk_Na+ = 2 weeks post 2nd salt treatment, 10wk_K = K+ 2 weeks post 2nd salt treatment, 10wk_Ca = Ca2+ 2 weeks post 2nd salt treatment.
The morphological and physiological traits comparisons were combined to produce a comparative outlook with the view to establish a clear picture on how the data correlates with different traits as presented in Figure 4.9.
Figure 4.9: A general correlation outlook using the whole data representing relationship between morphological and physiological traits in B. oleracea genotypes following salt treatment at different physiological stages of plant growth. Legend: +1 to -1 represent perfect positive correlation to negative correlation. Key: Morphological Traits: PH = Plant Height, LFW = Leaf Fresh Weight, LDW = Leaf Dry weight, LA = Leaf Area. Physiological Traits: 24hr_Na = Na+
24hr post 1st salt treatment, 24hr_K = K+ 24hr post 1st salt treatment, 24hr_Ca = Ca2+ 24hr post
1st salt treatment, 8wk_Na = Na+ 2 weeks post 1st salt treatment, 8wk_K = K+ 2 weeks post 1st
salt treatment, 8wk_Ca = Ca2+ 2 weeks post 1st salt treatment, 8wk24hr_Na = Na+ 24hr post 2nd
salt treatment, 8wk24hr_K = K+ 24hr post 2nd salt treatment, 8wk24hr_Ca = Ca2+ 24hr post 2nd
salt treatment, 10wk_Na+ = 2 weeks post 2nd salt treatment, 10wk_K = K+ 2 weeks post 2nd salt
treatment, 10wk_Ca = Ca2+ 2 weeks post 2nd salt treatment.
−1 −0.8 −0.6 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 LFW 8wk24hr_Na10wk_KPH 2wk pt_Na10wk_NaLA 2wk pt_Ca8wk24hr_Ca24hr_Ca24hr_Na24hr_K2wk pt_KLDW8wk24hr_K
LFW 8wk24hr_Na 10wk_K PH 2wk pt_Na 10wk_Na LA 2wk pt_Ca 8wk24hr_Ca 24hr_Ca 24hr_Na 24hr_K 2wk pt_K LDW 8wk24hr_K
Principal component (PC) analysis was carried out on the data to determine the proportion of variances retained by the principal components. This was done by extracting the eigenvalues, which correspond to the amount of the variation explained by each principal component as presented in Figure 4.10.
Figure 4.10: A plot of eigenvalues and variance associated with components generated from the data. 1 2 3 4 5 6 7 8 Variance Principal Components Percentage of v ar iance 0 5 10 15 20 25 30 35
Figure 4.11: The strength of variables contribution in the formation of PC plot. Legend: light blue to dark blue indicate variable contribution from higher to lower. Key: Morphological Traits: PH = Plant Height, LFW = Leaf Fresh Weight, LDW = Leaf Dry weight, LA = Leaf Area. Physiological Traits: 24hr_Na = Na+ 24hr post 1st salt treatment, 24hr_K = K+ 24hr post 1st salt
treatment, 24hr_Ca = Ca2+ 24hr post 1st salt treatment, 8wk_Na = Na+ 2 weeks post 1st salt
treatment, 8wk_K = K+ 2 weeks post 1st salt treatment, 8wk_Ca = Ca2+ 2 weeks post 1st salt
treatment, 8wk24hr_Na = Na+ 24hr post 2nd salt treatment, 8wk24hr_K = K+ 24hr post 2nd salt
treatment, 8wk24hr_Ca = Ca2+ 24hr post 2nd salt treatment, 10wk_Na+ = 2 weeks post 2nd salt
treatment, 10wk_K = K+ 2 weeks post 2nd salt treatment, 10wk_Ca = Ca2+ 2 weeks post 2nd salt
treatment. PH LFW LDW LA 24hr_Na 2wk pt_Na 8wk24hr_Na 10wk_Na 24hr_K 2wk pt_K 8wk24hr_K 10wk_K 24hr_Ca 2wk pt_Ca 8wk24hr_Ca −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 Dim1 (36.4%) Dim2 (20.9%) 2.5 5.0 7.5 10.0 contrib Variables − PCA
A biplot was also plotted to group the variables measured (the morphological and physiological traits) together with B. oleracea lines to enable draw relationships between the B. oleracea lines based on variables. Figure 4.12.
Figure 4.12: A biplot of variables and individual B. oleracea genotypes. Note: 1 = the founder rapid cycling line DHSL150, 2 = B. bourgaei-S1 (C07007), 3 = C10025-DH, 4 = C13013-DH,
5 = C13001-DH, 6 = B. oleracea-S1 (C07060), 7 = C10128-DH, 8 = B. oleracea-S1(C07079A) and 9 = C10121-DH. Key: Morphological Traits: PH = Plant Height, LFW = Leaf Fresh Weight, LDW = Leaf Dry weight, LA = Leaf Area. Physiological Traits: 24hr_Na = Na+ 24hr post 1st
salt treatment, 24hr_K = K+ 24hr post 1st salt treatment, 24hr_Ca = Ca2+ 24hr post 1st salt
treatment, 8wk_Na = Na+ 2 weeks post 1st salt treatment, 8wk_K = K+ 2 weeks post 1st salt
treatment, 8wk_Ca = Ca2+ 2 weeks post 1st salt treatment, 8wk24hr_Na = Na+ 24hr post 2nd salt
treatment, 8wk24hr_K = K+ 24hr post 2nd salt treatment, 8wk24hr_Ca = Ca2+ 24hr post 2nd salt
treatment, 10wk_Na+ = 2 weeks post 2nd salt treatment, 10wk_K = K+ 2 weeks post 2nd salt
treatment, 10wk_Ca = Ca2+ 2 weeks post 2nd salt treatment.
1 2 3 4 5 6 7 8 9 PH LFW LDW LA 24hr_Na 2wk pt_Na 8wk24hr_Na 10wk_Na 24hr_K 2wk pt_K 8wk24hr_K 10wk_K 24hr_Ca 2wk pt_Ca 8wk24hr_Ca −2 0 2 −4 −2 0 2 4 Dim1 (36.4%) Dim2 (20.9%) PCA − Biplot
4.3 Discussion
Plants exposed to higher salt experience stress, both hyper-ionic and hyperosmotic through an accumulation of Na+ and Cl- ions, which causes membrane damage, nutrient imbalance,
enzymatic inhibition, metabolic dysfunction, etc. The quick and basic response of plants exposed to higher salt has been K+ efflux from the cells caused by excess Na+ (Nedjimi and
Daoud, 2009; Anschutz et al., 2014; Bose et al., 2014b). The huge Na+ influx in the plant
growth medium creates a plasma membrane depolarisation which further activates membrane-bound cation channels, the guard cell outward rectifying potassium channels (GORK), that stimulate Na+ diffusion into the cell, and K+ efflux and thereby increasing
Na+ content (Blumwald et al., 2000; Demidchik and Tester, 2002). Salt-induced stress
creates disruption of the K+/Na+ ratio (Simaei et al., 2012) and interferes with K+
homeostasis (TunVtürk et al., 2008).
From our result, we observed that salt stress-induced causes an increase in Na+ leaf content
twenty-four hours post-treatment within the B. oleracea genotypes. Increase in Na+ leaf
content following salt stress has been reported in many studies by Essa (2002); Yasar et al. (2006); Kusvuran et al. (2007) and work on canola genotypes by TunVtürk et al. (2011). The level of potassium was affected by two-weeks post-treatment. Reduction in potassium level due salt stress has been widely reported e.g., studies by Essa (2002); Ashraf and McNeilly (2004); Li et al. (2006); Yasar et al. (2006); Bandeh-Hagh et al. (2008); Rahman et al. (2016). Potassium plays a critical role in the neutralisation reactions of anion and regulates cell membrane polarisation, osmoregulation, likewise being an important factor in the activity of enzymes involved in many metabolic pathways (Very et al., 2014). The reduction of potassium level was attributed to the entry of the higher amount of Na+ into plant roots cell by non-selective cation channels (NSCC) that cause K+ efflux or leakage through guard
cell outward rectifying potassium channel (GORK) and stellar K+ outward rectifying
channel (SKOR) (Rahman et al., 2016). Other potassium transporters that might be implicated are membrane-bound protein channels actively involved in transportation of potassium like Shaker K+ channel, High affinity potassium (HAK), potassium uptake
(KUP), potassium transporter (KT) and high affinity potassium transporter (HKT) amongst others (Wang and Wu, 2013; Very et al., 2014; Shabala and Pottosin, 2014).
Higher Na+ accumulation was observed two-weeks post-treatment leading to a reduction of
K+/Na+ ratio, which might be as a result of disruption of ion homeostasis. The Na+ influx
and K+ efflux have been associated with increased ROS production that could lead to the
activation of NSCC (Maathius, 2006). A similar report showed that higher Na+ lead to the disruption of ion homeostasis under salt stress conditions (TunVtürk et al., 2008; Wu and Wang, 2012). From our result, Ca2+ level showed to be relatively unaffected in Brassica
oleracea genotypes despite higher Na+ in the growth medium. Moreover, it has been
reported that exogenous calcium promotes membrane stability, thus ameliorating salt toxicity by decreasing Na+ influx through NSCC and indirectly inhibiting K+ efflux through
GORK channel in plants (Cramer et al., 1985; Essa et al., 2003; Shabala et al., 2006; Nedjimi and Daoud, 2009; Shabala and Pottosin, 2014). More so, exogenous calcium has shown to cause a reduction in the uptake and transport of Na+ and further preventing it from binding
to the cell wall (Kurth et al., 1986; Rubio et al., 2003). Other functions of cellular and vacuolar calcium include blockage of the fast vacuole (FV) channel in a voltage-dependent and independent reaction preventing Na+ from being leak back into vacuole and ultimately
Improved Ca2+ level observed in B. oleracea genotypes might be the cause of improved K+
level observed after the plants were salt stress for the second time thus, leading to higher K+/Na+ ratio. Although an increase in Na+ leaf content was observed, the B. oleracea
genotypes were able to improve and retained their Ca2+ level unaffected. Ca2+ ion, in
optimum concentrations, has been shown to play crucial roles in many physiological processes of plants and shown to increase plant resistance to abiotic stresses (Watkin et al., 2000). In addition, it has been suggested that Ca2+ participates in the regulatory mechanism
of plants’ adjustment to adverse conditions; high temperature, cold injury, drought stress and salt stress (Arora and Palta, 1989; Bowler and Fluhr, 2000; Mozafari et al., 2008; Joshi et al., 2012). It was reported also that higher extracellular Ca2+/Na+ ratio causes a reduction
4.4 Summary
Salt stress triggers a plant’s physiological and morphological response for adaptation and growth. This chapter investigates the physiological variation in B. oleracea genotypes exposed to salt stress using 250 mM NaCl. The result demonstrates significant variation between the parent lines; DHSL150, the wild S1, and their derived DH lines. The level of different nutrients content; Na+, K+, and Ca2+ analysed have shown significant variation.
Leaf Na+ content rises 24 hr post-treatment in all the B. oleracea lines, indicating
physiological response related to an osmotic phase of salt stress whereby Na+ influxes causes cell-water imbalances. It also shows a delay in activation of Na+ exclusion channels
that shown to recycle Na+ in and out, so as to reduce its accumulation in the plant cell further
preventing it from reaching the important plant organs such as leaves. Furthermore, it is an indication that the B. oleracea lines share similar biochemical response in handling Na+ 24
hr post-treatment. Potassium (K+), an osmotically important element in plant cell plays a
critical role in both structure and maintenance of cell turgor has shown to be relatively unaffected 24 hr post-treatment, so also the Ca2+ level, which remains unchanged. This
chapter also reported the deteriorating effects of excess salt two-weeks post-treatment on K+ level, an indication of homeostasis effects of Na+ against K+, which is widely reported
in many reviews that Na+ competes with K+ thereby displaces it to get into the cell especially
through non-selective cation channels (SNCCs) and other low affinity K+ channels such as
K+ outward-rectifying (KOR) channels and inward-rectifying (KIR) channels as a result of
membrane depolarisation by excess Na+, which led to movement of Na+ and reduction of
K+. These processes could be the cause of K+ reduction and increase Na+ observed in the B.
oleracea lines. Although significant variation observed between genotypes, a statistical significant reduction was observed in the cultivated rapid cycling DHSL150 and a DH line (C13013) as against their untreated control lines while in wild S1 lines, and four other DH
lines have shown non-significant reduction as compared to their control lines. This could also be one of the reasons of high Na+/K+ ratio observed in these lines and high cellular
Na+/K+ ratio is attributed salt resilience in many plants especially the salt tolerant ones.
Different correlation analysis have been conducted to establish a relationship between the observed morphological variation and physiological response, different traits have shown both strong positive and negative correlation based on individual lines and this was further corroborated when the data was put together to creates to general outlook which confirms the individual correlations that the morphological variation observed is connected to physiological response. Principal component analysis conducted further group the lines based on the quality of variable contribution in PC generation and how it relates to individual lines where some DH lines were put together with the wild S1 parent in one quadrant of PC biplot. Signifies good morphological and physiological relationship in response to salt stress in these B. oleracea lines and both wild S1 and some DH lines have shown better performance to salt stress than the cultivated rapid cycling DHSL150. However, more work is recommended to enable their further classification based on salt resilience.
CHAPTER FIVE
Relative Gene Expression of Ion Membrane Transporters in B.
5.0 Introduction
As described in Chapter One, salt tolerance within plants involves reduction of excess Na+ and retention of K+ ions in the cytosol. This has been attributed to be regulated by the
membrane ion transporters. This are involved in either removing the added Na+ from cells through a membrane-bound Na+/H+ antiporter, and or via sequestration of excess Na+ in
vacuoles through the NHX or Na+/H+ antiporter in the vacuole membrane (Blumwald and
Pool, 1985; Zhu, 2003; Bassil et al., 2012). These membrane transporters are also involved in K+ sequestration within the vacuole. Both types are ubiquitous membrane proteins that