Full Thesis is on Research Gate DOI:10.13140/RG.2.2.14708.63368
CHAPTER 6
GENERAL DISCUSSION
6.1 Sun and shades characteristics of rice leaves
Leaves that grow in either high light or low light environments are known to be different in terms of leaf thickness (Terashima et al., 2001), chloroplast structure (Lichtenthaler, et al., 1981), pigment contents (Anderson et al., 1995) and assimilation rates (Boardman, 1977). The initial aim of the work reported here was to characterise sun and shade type leaves in rice with a special emphasis on stomatal properties. Previous work on the effects of irradiance level on stomata and photosynthesis were generally conducted using Arabidopsis (Coupe et al., 2006; Bussis et al., 2006) with more limited analysis of grass leaves, such as rice. A deeper understanding of stomatal development in rice and its relationship to leaf function under different environmental conditions will provide a better understanding of rice leaf function.
In rice, guard cells are flanked by a pair of subsidiary cells, thus producing a stomatal complex (Fig. 3.6). With respect to length and width properties, many size measurements can be derived from this stomatal complex structure but for the sun-shade comparison, only stomatal complex and pore areas (SCA and SPA respectively) were analysed. Approximation of pore area was obtained using a formula for elongated hexagon and its derivation has been described in section 2.3.3.3. Other studies such as Dow et al. (2014) assume pore area is an ellipse, which is reasonable for Arabidopsis guard cells which are kidney-shaped but in rice they are dumbbell-shaped, thus a long hexagon is proposed to be more appropriate for the area assessment (Fig. 2.2). This pore area formula for rice might be useful for other related grass crops such as maize and wheat.
HL conditions in general produced larger stomatal dimensions compared to LL grown leaves, thus supporting other published work on rice (Hubbart et al., 2012) and Arabidopsis (Coupe et al., 2005). However this is not a universal phenomenon since other plant species, such as tobacco, produce stomata of similar size in either high or low irradiance level (Thomas et al., 2003). SCA and SPA in HL grown rice are generally bigger in interveinal gaps (IGs) across the leaf width from midrib to the leaf margin (Fig. 3.5 A and B) compared to LL leaves, thus confirming previous work on rice (Hubbart et al., 2012). This detailed way of measuring stomata reveals that there is a tendency for stomata to be big (for HL leaves) in the first IG (immediately next to midrib) and the last IGs close to the leaf margin. This is more pronounced for SPA measurements. Middle IGs further away from midrib and margin seem to show more representative values for the entire leaf width, thus were the region chosen for analyses reported in chapter 4. The data reported here indicate that the sampling method does matter in stomatal studies in grasses such as rice. Care must be taken where stomata are sampled with respect to veins and leaf margin.
In general stomatal density (SD) was not different between HL and LL leaves across all IGs, although the last IG (in HL) certainly showed a higher SD than LL leaves (Fig. 3.5 C). This comparison scheme is purely based on an ‘IG to IG’ basis and since HL and LL produce different number of IGs in total, the last IG in HL is actually the 3rd last IG in LL, i.e. is not equivalent. As pointed out earlier, it is important to sample stomata in the most representative IGs, usually in the middle if the leaf width, and this approach has been explained in Fig. 3.8A and 3.8B. Besides SD, it is also possible to express patterning in rice using stomatal file percentage (%SF). Rice stomata only occur in certain epidermal files bound by two veins. %SF can vary because the number of “pure” epidermal cell files (without stomata) is not fixed and this parameter is used in Chapter 4 for further patterning analysis. In this work it is noticeable to see how in rice in almost all the leaves, independent of any treatments given, the stomatal file number (epidermal file that contains at least one stoma) varied only from two to four. This ‘maximum four stomatal files’ rule is only violated when the IG is next to the mid vein (Fig. 6.1 A) and close to the leaf margin (Fig. 6.1 C), otherwise the rule is true in all other IGs (Fig. 6.1 B).
Gas exchange analysis revealed that, as expected, HL leaves consistently had higher assimilation rates compared to LL leaves under varying CO2 levels, reflecting the expected sun-type photosynthetic apparatus and agreeing with many previously reported works (Murchie et al., 2005; Hubbart et al., 2013; Narawatthana, 2013). Especially at high CO2 levels in the A-Ci experiments, the carboxylation activity of Rubisco is highly elevated. Increasing CO2 concentration around Rubisco by 1000 ppm (and beyond) would almost eliminate all oxygenase activity (von Caemmerer, 2000), explaining the invariably higher assimilation rates obtained than under ambient CO2 level (around 400ppm).
However the curve fitting tool used in this study only focuses on the two primary limitations, namely Vcmax and Jmax. In the second phase of the curve the assimilation data points usually stay relatively flat, but sometimes when the Ca given is high enough, they can drop, as seen in HL leaves (Fig. 3.9). This phenomenon can be associated with the third phase of the curve, namely triose-phosphate utilization limitation (TPU) (von Caemmerer, 2000; Long and Bernacchi, 2003). Favouring carboxylation over oxygenase activity undoubtedly enhances carbon assimilation, which in turn produces a lot of 3-carbon sugars, namely triose-phosphate (3-phosphoglyceraldehyde). This is utilized either by transporting triose-phosphate from the chloroplast to cytosol or utilized in the chloroplast (Leegood, 1996). In the chloroplast, triose-phosphate is converted to starch, thus releasing the inorganic phosphate groups that can be reutilized in photophosphorylation (producing ATP needed to regenerate RuBP) (Sharkey, 1985; Leegood and Furbank, 1986). Thus, when TPU takes place other processes are affected, leading to a plateau in assimilation or even a decrease (in the case discussed here). This could be a mechanism employed to maintain photosynthesis to an optimum level (Sharkey et al., 2004) within the given overall HL leaf characteristics.
Stomatal conductance (gs) measured in these experiments showed that LL grown leaves have higher gs values compared to HL leaves in both oxygen conditions. This is a perplexing result because a number of works using rice (Narawatthana, 2013) and other species (Gross et al., 2008; Guidi et al., 2008) reported that sun leaves tend to have higher gs than shade leaves. Experimental set up could be the source of discrepancies, such as light and relative humidity levels. However, based on the gs modelling using a formula by Franks and Farquhar (2001), high gs is expected when stomata are relatively smaller (thus leading to higher density). But as discussed earlier, stomatal density is similar between HL and LL leaves, thus leaving the high gs to be explained by the third component of the formula, namely stomatal pore depth (SPD). I do not have the measurements for this parameter but there might be a good connection between SPD and general leaf thickness because the thin LL leaves (Narawatthana, 2013) may also have a relatively shallow SPD that facilitates gas movement. The difference of gs between HL and LL leaves could also possibly be explained by differential stomatal opening. If each stoma had a different capacity in controlling aperture due to its location (with respect to the vein) or natural solute contents, the cumulative effects could lead to the unusual varying gs responses reported in this study, which opens an opportunity for further investigation.
Figure 6.1:
Relative positions of stomatal files (S) with respect to midvein (MV), small vein (SV) or leaf margin (M). There are usually more than four stomatal files in the interveinal gap (IG) next to a MV (A). In IGs in the middle of the leaf width (B), stomatal files always follow the ‘maximum four rule’ The last IG next to margin (C) usually has only one stomatal file and a high in stomatal density.
Pigment quantification revealed that rice leaves follow the general view that HL leaves contain a higher chlorophyll a/b ratio (Kitajima and Hogan, 2003) than LL leaves because of relatively lower chlorophyll-b content (Fig. 3.12G). LL leaves invest more in making chlorophyll-b (hence more LHCII as well) as a predominant pigment molecule to capture and channel quantum energies. As showed by Evans and Seeman (1989), in terms of leaf nitrogen partitioning, sun and shade leaves invest almost similar amount of N to make light harvesting components, but HL leaves spend more N to make enzymes associated with carbon reduction, thus indicating LL leaves commitment to capture photons energy in shady environments. However it is worth noting that a number of Amazonian tree species have been shown to do the opposite by investing more in chlorophyll-a (Morais et al., 2007). Other photosynthetic organisms, such as diatoms and brown algae, are also known not to invest in chlorophyll-b (Northrop and Connor, 2008) under similar conditions.
Besides being photosynthetically active, HL leaves are also efficient at managing water, made evident by a higher intrinsic water use efficiency (iWUE) than LL leaves (Fig. 3.13A) The analysis of carbon isotope ratio also showed that HL leaves had more positive values, thus showing a higher discrimination against the heavier 13C compared to LL leaves (Fig. 3.13B). This happens when CO2 demand at the carboxylation site is relatively high due to the abundance and activity of Rubisco. During such a situation Rubisco becomes less stringent in assimilating CO2 from the heavier 13C isotope, although in normal conditions the lighter 12C is always the choice. Stomatal aperture can also be minimal since the rapid drawdown at the carboxylation site creates a strong gradient drives CO2 influx, thus resulting in lower gs. This is translated into a higher water use efficiency (Franks et al., 2015), which is a desirable quality in crop plants.
6.2 Leaf development, stomatal patterning and leaf performance
Stomatal differentiation occurs during relatively early stages of leaf development, yet this pattern of stomata is likely to significantly influence the photosynthetic performance of the mature leaf. Plants have thus evolved mechanisms by which stomatal patterning in the early phase of leaf development is attuned to the prevailing environmental conditions, including light. A second part of this thesis aimed to investigate the relationship of leaf developmental stage to the ability to respond to the light environment by altering aspects of stomatal differentiation.
To perform such an investigation one first has to have a robust means of assigning leaf developmental stage. It has been 6 decades since Ralph Erickson and Francis Michelini proposed the idea of plastochron index for studying plant stem development (Erickson and Michelini, 1957). Historically, the plastochron itself is not a new concept since the term was coined by Askenasy in 1880, referring to the elapsed time between initiation of two successive internode cells of Nitella algae. Esau (Esau, 1965) highlighted that this general definition by Askenasy could be applied to leaf primordium initiation, apical growth, internodes or axillary buds or floral organs development, as well as similar stages of shoot vascularization. Thus in terms of leaf development Schmidt (1924) was regarded by Esau as the first author to specifically used plastochron as the period of time between the initiation of two successive individual, pairs, or whorls of leaf primordia by shoot apical meristem (SAM).
In the first part of chapter 4, I established a plastochron index system for rice which allowed me to robustly assign leaf developmental stage. Importantly, due to the robust patterning of leaf initiation over time, this system allowed me to accurately assess the developmental stage of very young primordia (hidden from view), thus facilitating transfer experiments where leaf primordia at specific stages of development were transferred from one light regime to another. Thus, a series of transfer experiments were performed on rice growing initially under HL or LL conditions. When the leaf no. 5 (which is hidden in layers of outer leaf sheath) had reached plastochron (P) stage 1,3 or 5 (using leaf no. 3 length as a proxy for estimation) the plant was transferred to the opposite light condition. The idea behind this experiment was that although leaf no. 5 is not directly exposed to the new light condition, it can still undergo acclimation to adjust the developing leaf to the new light condition by means of systemic signalling originating from mature leaves (Fig. 6.2). There are a number of mobile signals that can be transduced via the vascular system from mature leaves to the developing primordia such as sugars, peptides and phytohormones (Coupe et al., 2006; Yoshida et al., 2011). The focus of this investigation was on the leaf response to the systemic signal rather than the signal itself, the nature of which remains a mystery and which deserves further investigation. With a prolonged exposure of the plant to a new light condition, leaf no. 5 is expected to adapt both physiologically and morphologically, including changes to stomatal phenotype (a major focus of this investigation). The main question was whether the developmental stage of leaf 5 had a major influence on the ability of the leaf to respond to these systemic signals.
In the transfer experiments reported here, the primordia at stages P1 and P3 generally showed an ability to respond to exposure of the plant to a new light regime by altering aspects of epidermal patterning, whereas by the P5 stage this was lost. Even in some parameters not showing a significant shift in mean value, there was a significant difference in variation when transfer was performed at the early (P1, P3) stages of leaf development. Taken together, these data indicate that the epidermis of rice leaves retains a significant plasticity up to the P3 stage, but that this plasticity is lost by the P5 stage of development. There thus appears to be a sensitivity window for rice leaves to alter stomatal phenotypes in response to altered new light conditions. Published data from our group suggests that various photosynthetic-related properties are also alterable in these P-stages (Van Campen et al., 2016). For both dicot (Gonzalez et al., 2012) and monocot (Fournier et al., 2005) leaves, virtually all cells are actively dividing at the beginning of leaf primordia development from the SAM. As development advances, cells at the leaf apex stop dividing and starts expanding while cells at the base are still in a phase of division. It is worth nothing that low light extends the duration of cell production, particularly in grass species growing with an abundance of nutrients (Sugiyama and Gotoh, 2010). Therefore in the transfer experiments from HL to LL conditions, it is expected that the number of epidermal cells will increase, consistent with the since P1 and P3-transferred leaves having the highest values for cell file number (Fig. 4.3F). However the increased number of dividing cells under LL conditions also tends to come with higher variation in cell number, as reported by Sugiyama and Gotoh (2010) using Festuca arundinacea, and a similar trend is reported here with the P1 and P3-transferred leaves. Thus the high variation in some parameters following transfer at early P stages to LL conditions might be explained by an increased rate of cell division. The higher number of cells which have the potential to undergo differentiation to, for example, stomata, might simply lead to more noise in the system, thus higher variation.
Figure 6.2:
Schematic illustration of potential systemic signaling relay system via vascular system perceived by older leaves that influence leaf acclimatization in developing and concealed leaf primordium.
It is interesting that transfer from LL to HL conditions generally led to less dramatic shifts in patterns of epidermal characteristics (Fig. 4.5). This might relate to the mechanism of systemic signalling from mature to young leaves which is thought to underpin the regulation of epidermal properties in the developing leaf primordia. The nature of this signal is unknown, but if it is related to metabolic/photosynthetic activity in the mature leaves (Hou et al., 2015) it might be that the shift to 750 µmol m-2 s-1 (HL under our conditions) was not sufficient to generate a strong signal. A shift to higher irradiance (e.g., 1000 µmol m-2 s-1) might be required to trigger a stronger response., The nature of the systemic-signal generating mechanism remains a major unknown. Efforts are emerging in understanding Target of Rapamycin (TOR, a highly conserved protein kinase) signaling network in multicellular plants (Xiong and Sheen, 2015) and sugars derived from photosynthesis, such as sucrose, seems to be the most likely signals involved in activating plant TOR kinase (Ren et al., 2013; Xiong et al., 2013). Studies have revealed that the TOR pathway is an essential controller for cytoplasmic growth, cellular development and proliferation (Zhang et al., 2013; Sablowski and Dornelas, 2014). Future work might explore the role of TOR activity in the source leaf response to altered irradiance, as well as the potential role of TOR signaling in modulating the cellular response in the target leaves.
An interesting observation from the transfer experiments is that guard cell width is the only parameter that is significantly altered following any P-stage transfer. This degree of plasticity implies GCW is not especially restricted by stomatal complex width. Guard cells in rice are sandwiched between pair of subsidiary cells whose partial collapse is required for guard cells to swell. A deeper understanding of the functional relationship of the guard cells and subsidiary cells may reveal insight into the plasticity of grass (rice) stomatal response to environmental change (Lawson, 2009).
Although stomatal density in HL and LL leaves was similar there was a higher spread of data in the HL to LL transfer experiment, especially in the P1 and P3 transfers (Fig. 4.3I). This suggest that the rice epidermis can respond to the LL environment during these P-stages by initiating longitudinal cell divisions (resulting in higher number of cell files, Fig. 43F) and varying the cell file width (Fig. 4.3H). These cellular changes are associated with more irregular stomatal differentiation, resulting in the higher variation in the SD values obtained. Besides density, stomatal patterning in rice can also be expressed in terms of stomatal file percentage (%SF). Since the IG used for this study has been selected based on the scheme in Fig. 3.5A and 3.5B, the number of cell files that contain stomata within any single interveinal gap always lies between two and four (Fig. 6.1B). This property, combined with the leaf’s ability to change CFN and CFW, leads to a significantly higher percentage of cell files containing stomata in HL grown leaves compared to LL grown leaves. This %SF parameter follows the trend in which P3 transfer appears to be the cut-off point to produce significant alteration in final stomatal properties in the mature leaf in response to altered irradiance. These data thus fit to a general cell division response being possible in P1 and P3 stages but not in P5 leaves, i.e., the systemic signalling system is not stomata specific but may alter the entire field of cells within which the stomata patterning system functions.
Since the experiments summarised above led to changes in stomatal properties, I investigated the extent to which this led to altered photosynthetic performance in the target leaves. To do this, a paired experiment was performed in which measurements of physiological parameters such as assimilation rate (A), stomatal conductance (gs) and intrinsic water use efficiency (iWUE) were associated with the stomatal phenotypes observed in the same leaf.
In the transfer experiment (HL to LL condition), transfer at the latest P5 stage, marked by the first appearance of the leaf blade from the sheath, conferred no significant changes in the stomatal parameters measured. However even there was no change in the stomatal parameters at maturity (P6-stage) in the LL condition, the relatively long period of time (5-6 days) from P5 to P6 allowed the leaves to acclimate physiologically (Fig. 4.16iv). However, the acclimation was only partial since the leaves only achieved Amax values similar to LL grown leaves after transfer at any P-stage (Fig. 4.7B) whereas A400 in general followed the trend where transfer at P1 and/or P3 was the cut-off point to produce leaves with an assimilation rate similar to LL grown leaves (Fig. 4.7A). Among the photosynthetic components analysed, only Vcmax of the P-transferred leaves showed comparable values to the LL grown leaves (Fig. 4.7C), suggesting a leaf’s ability to change Rubisco content continues to a relatively later developmental stage.
The opposite transfer from LL to HL conditions generally revealed less plasticity in the stomatal parameters measured (Fig. 4.5B). However the measured A400 and Amax values for the P-5 transferred leaves were similar to HL grown leaves (Fig 4.9A and B). Analysis also revealed that leaves transferred at any P-stages were able to increase Vcmax (Fig 4.9C), Jmax (Fig. 4.9E) and chlorophyll a/b ratio (4.9F) to match those observed in HL grown leaves. Taking all the physiological data together, it seems that stomata (despite not changing much either in size or density) are able to ‘work more’ under some conditions, i.e., they can open more to allow for more CO2 diffusion, thus allowing an increase in photosynthesis. This is possible since the measured (operational) gs results from partially open stomatal aperture. When the need arises (e.g, low CO2 levels, high RH, blue-red light and possibly high photosynthetic capacity, as in this study), stomata have the capacity to increase their aperture (Dow et al., 2014), thus overriding their normal, submaximal aperture size.
The analysis of gs-Ci curves also revealed some interesting findings about gs responses in rice. In this entire study rice was always grown hydroponically, therefore access to water was never an issue. Thus, theoretically, when CO2 is steadily increasing from low concentrations, stomata should open to allow more rapid gas exchange without any danger of increased transpiration having a negative outcome on their ability to maintain turgor pressure and water potential. However in all the gs-Ci curves analyzed, stomatal closing occurred before ambient CO2 level (400ppm). This tight gs response seems to be pre-programmed, indicating the ability of rice to maintain optimum water use efficiency in any given water status condition.
Ci concentrations in leaves have long been known to regulate gs (Mott, 1988) and are are determined by cellular respiration, photosynthesis rate and atmospheric CO2 levels (Ca) (Lawson et al., 2014). However the role of photosynthesis in gs regulation has been a matter of debate for many years since two photosynthetic cells, namely mesophyll (via mesophyll-derived signals, Mott et al. (2014)) and guard cells (via osmotic adjustment, Lawson et al., (2003); Lawson et al., (2014)) make contributions to stomatal aperture control in response to CO2 changes. However recent findings have shown that starch biosynthesis in guard cells is required for proper stomatal closure under high Ca condition, while starch biosynthesis in mesophyll cells makes a negligible contribution to the process (Azoulay-Shemer et al., 2016). Moreover elevated CO2 levels also enhance anion channel activity in guard cells, inducing stomatal closure via the action of the SLAC1 protein that regulates gates for anion transport (Yamamoto et al., 2016). But why did stomatal closure always occur before the ambient CO2 level in this present study? As mentioned, starch accumulation in guard cells is required for stomatal closure. During the increase in Ca levels, photosynthesis rate increases progressively so that where sugars such as sucrose, fructose and glucose accumulate, helping to drive stomatal opening (Antunes et al., 2012). Sugar accumulation, at sub-ambient Ca level, leads to conversion to starch that which helps drive stomatal closure. In addition, there is no need to widely open stomata at high Ca levels since CO2 will readily diffuse across the concentration gradient into substomatal chamber. The plant thus has a variety of mechanism to achieve optimal balance between CO2 uptake and water-loss over a range of ideal and sub-optimal growth conditions (Haworth et al.,2015). A recent finding has confirmed this, showing that plants grown under elevated CO2 condition produce less responsive stomata (poor aperture control), causing the leaves to become more vulnerable to water stress and high temperature (Haworth et al., 2016).
Among all the physiological parameters analysed in this study, iWUE at ambient CO2 is the only one that showed significant correlation with one stomatal phenotype, namely guard cell width. As discussed earlier, it is noteworthy that GCW is the only parameter that significantly altered in the generally less responsive LL-HL transfer experiment. It seems that GCW is also indicative of the plants iWUE. This is consistent with the widely observed importance of guard cells in stomatal control, which in turn affects general leaf performance. (Lawson and Blatt, 2014). Although the mechanistic link of GCW and iWUE is not entirely clear, this information could be exploited by crop breeders in the search for useful traits for drought prone areas or for general water conservation in agricultural crops. Effective stomatal control is crucial to the plant stress response (Killi et al.,2017) and the identification of any physical parameter which could be used as a proxy for iWUE would be very useful in screening programs.
6.3 Optimization of a tool for studying gene expression in rice
In the final part of this thesis I developed a technique with the ultimate aim to examine the expression of genes involved in stomatal differentiation and how these might respond to altered environment. The whole-mount in situ hybridisation (WISH) technique was chosen as a tool to study gene expression in rice. Due to time limitations I could not prepare the probes for stomata-related genes but was fortunate enough to obtain probes for various genes from colleagues which were used to optimise the procedure. WISH is a very useful tool in studying gene expression because it saves time and is less laborious compared to the commonly used in situ hybridization technique that involves embedding and sectioning of biological tissues.
WISH in this study was based on the protocol by Traas (2008) who optimized it for use on young, soft Arabidopsis, such as embryos and leaf primordia. Without any modification of the original protocol, hybridisation of the control probe (for the eEF1A gene) already yielded promising results, especially since the bulliform cells were completely stain-free with the antisense probes (Fig. 5.3). However it was still not perfect because some background and non-specific staining also occurred in some interveinal gap areas and x-shaped silica bodies. I then performed an intensive optimization of WISH for rice, starting by formulating a blocker concoction to prevent non-specific interactions of antibody with the tissue that can lead to false staining. The results showed that no single protein mixture could be a universal blocker because I had to combine three reagents, namely bovine albumin serum, sheep serum and antibody pre-adsorbed to acetone powder, in order to obtain a satisfactorily clear and stain-free leaf tissue indicating the absence of non-specific antibody binding (Fig 5.5). Further optimization was also made in terms of tissue preparation by making a transverse cut on intact leaf clusters to promote tissue penetration. At the same time this sectioning approach allowed signal detection in leaves of different ages which have distinct structures associated with them due to their different maturity levels, such as undifferentiated epidermal cells, lignified dermal layers, vascular systems and lacuna (Fig.5.2). Staining time is also an important factor in WISH, so for each probe tested the enzymatic reaction in the colour development was stopped as soon as sufficient signal was detected. This combined WISH reagent optimization and transverse cutting are termed WISH-TC.
Comparison of the results reported here with other work on similar genes shows that WISH-TC manages to produce a highly comparable result, suggesting that it is a reliable technique to be used on other probes. These highly similar results included probes for the genes Histone-H4 (Fig. 5.7: compare with Van Campen, unpublished) and DROOPING LEAF (Fig. 5.9: compare with Ohmori et al., 2011) where both comparisons are with rice. Some probes compared against data from Arabidopsis (a different species) still yielded positive and similar gene expression patterns, such as probes for the genes MONOPTEROS (Fig. 5.10: Compared Wenzel et al., 2007) and DELTA-7-STEROL-C5-DESATURASE (DWF7) (Fig. 5.12: compare with Silvestro, 2013). Some of these genes are either putative or of unknown functions in rice, such as DWF7 and CUL1. It is interesting to see DW7 expression is concentrated around the edges of lacuna since in Arabidopsis it is linked to oleosin formation, thus if oleosin is present in rice this could mean it is involved in the desiccation of lacuna regions (Hsieh and Huang, 2007). While CUL1 protein function is putative in rice, in Arabidopsis its role is known to be a component of a protein complex, namely CULLIN-RING E3 ubiquitin ligase complex which controls many aspects of plant development (Hua and Vierstra, 2011). Observation of CUL1 expression in rice using WISH-TC suggests concentration around lacuna edges (like DW7) which could lead to a functional interpretation via more confirmative work.
In this study WISH-TC has been shown to work in a reliable fashion but it is still not a universal method since it sometimes gives data which are uncertain and difficult to interpret, for example with the THYLAKOID FORMATION FACTOR (THF1) and DELTA-14-STEROL REDUCTASE (FACKEL) probes (Fig. 5.15 and 5.16). This suggests further optimization has to take place with certain probes, including probe hydrolysis (ensuring probe length <500bp to promote tissue penetration), the use of additional protein blockers such as non-fat dry milk powder, and possibly further tissue digestion to promote probe penetration using cellulase enzymes which can be particularly useful in complex tissue (Rozier et al., 2014). Staining time and ambient temperature are also very important because, as mentioned earlier, the colour develops enzymatically. A constant ambient temperature would certainly be helpful in giving more power to be precise in stopping the reactions once sufficient signals have been detected for the antisense probes. For future development of WISH it is advisable to have an incubator so that temperature can be kept constant at all stages of the experiment, especially providing more reliability in the reaction time during the final staining step.
6.4 Concluding remarks and future work
Very early stages in rice leaf development (up to P3) have been identified as a very dynamic phase in which the leaf primordium is responsive to the systemic signals generated in the mature leaves which indicate the overall light regime of the plant. The transfer from HL to LL indicated that the rice leaf is more responsive in terms of physical and physiological acclimation for this transfer compared to transfer from LL to HL. Acclimatisation during the P3 stage is a co-ordinated processes involving stomatal differentiation and biochemical alteration to better prepare the leaf to the current light conditions. As pointed out by Van Campen et al (2016), stages prior to P4 can be manipulated since photosynthetic competence is being established during this early stage. Even though transfer at the late P5 stage is insufficient to confer significant stomatal changes, the time from P5 to maturity (about 5 days) is enough for the leaf to alter the physiological photosynthetic machinery, thus compensating for limited stomatal adaptation. It is interesting that the guard cells width (GCW) parameter kept cropping up as the most plastic parameter during the transfer experiments, and in the paired experiments of stomatal structure and leaf physiology. It is the only stomatal parameter that had significant correlation with leaf water use efficiency.
The optimization of the WISH method for rice has shown reliability and superiority in terms of time and general workload compared to the traditional in situ hybridisation method. As has been pointed out earlier, the main eventual aim of this technique optimisation was to study the control of stomatal development in rice, but due to long time taken for optimization it could only be performed using the available control probes. This work certainly opens up the opportunity to try WISH using stomatal related genes such as OsSPCH, OsMute and OsFama. The level of mRNA for these genes might be low so further optimization on the WISH method might be needed. Future work might include the use of fluorimetric WISH instead of the regular colorimetric staining so that multiple probes labelled with different fluorescent signals could be introduced at the same time, yielding a more comprehensive picture regarding different gene expression and localization in the same tissue.
In conclusion, the work reported in this thesis provides an insight into the differentiation of stomata in rice, in particular how it responds to altered irradiance at particular stages of leaf development. Understanding the genetic mechanism by which this response occurs will provide a better understanding of how rice leaves adapt to the environment, which may help in the selection of plants for improved crop performance.
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influence on photosynthetic performance[Unpublished Doctoral dissertation]. The University of Sheffield
The control of stomatal properties in rice (Oryza sativa L.) and their influence on photosynthetic performance
June 2017
DOI:
Thesis for: Doctor of Philosophy
Advisor: Prof. Andrew J. Fleming
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