The Role of Guard Cells Chloroplasts toward the Enhancement of Plant Growth

Abstract

Guard cells contain chloroplasts, and the stomata through which exchange of gas takes place. They control the stomatal pore, which serves as a channel for exchange of gas by balancing between CO2 uptake for photosynthesis and water loss through transpiration. As a result, chloroplasts in the guard cells have become potential tool for manipulation toward improvement of plant productivity through photosynthesis. The role of the guard cells chloroplasts can, therefore, be elucidated through manipulations of enzymes for photosynthesis by using molecular means. The cytochrome b6f complex catalyzes the transfer of electrons between the two photosynthetic reaction centers, Photosystems II and Photosystem I, while at the same time, transferring protons across the thylakoid used to synthesize ATP for the Calvin cycle. In this study, the overexpression of the Rieske FeS protein in Arabidopsis exhibited phenotypes, which resulted in substantial improvements of quantum efficiency of PSII. Transgenic lines were significantly higher in early development of the plants. Phenotypes observed in the transformed plants also showed faster initial growth rates evidenced by larger leaf area and faster rosette increases, which may suggest that Rieske might be of importance for enhanced plant growth. The result obtained proves more opportunities await the exploitation of guard cells chloroplasts metabolism toward the improvement of plants.

1. Introduction

Stomatal conductance determines the flux of gases between the inside of the leaf and the external atmosphere, which influences photosynthetic carbon assimilation and water use. Understanding the structure, function, and signaling mechanism in stomata in response to changing environmental conditions is, therefore, critically important if we are to manipulate the processes for optimal plant use (Figure 1) [1, 2, 3].

Figure 1.

Guard cells, stomata, and chloroplasts as potential tools for manipulation. (A) Guard cells from an epidermal peel showing chloroplasts, which are the site of photosynthesis and the stomata through which exchange of gas takes place. The guard cells control the stomatal pore, that is. The opening and closing of the stomata. (B) Schematic diagram of a single chloroplast illustrating (a) the thylakoid membrane where electron transport chain takes place. The cytochrome b₆f between the PSII and PSI shuttles electrons, which are used for the synthesis of NADPH and ATP as energy for fueling the Calvin cycle. (b) Calvin cycle found in the stroma of the chloroplast where sedoheptulose-1, 7-bisphosphatase (SBPase) a key component in the regeneration of RuBP in the Calvin cycle functions. (source: Picture taken from PhD research of Batta kucheli 2018).

Stomata has, therefore, attracted the attention of scientists for almost three centuries [4], and a great deal of knowledge related to the structure, development, and physiology of stomata has been acquired. In order to balance between carbon supply and the ability to optimize or sustain plant growth in an ever-fluctuating environment, an understanding of the external and internal responses is required, as well as new approaches that integrate both molecular and physiological approaches [5, 6]. Genetic potentials for maximum yield still lie unrealized, and the need for better adaptation of plants to climatic factors in which the plants are grown are still enormous [7].

In view of this, this chapter highlights or provides a way to elucidate the role of guard cell chloroplasts by using molecular tools and techniques to manipulate chloroplast metabolism specifically in the guard cells.

1.1 Guard cell chloroplasts

Photosynthesis takes place primarily in the mesophyll tissue as epidermal cells generally lack chloroplasts. However, guard cells, which developed from protodermal cells, also contain photosynthetically active chloroplasts in most but not all species [8, 9, 10, 11, 12]. Guard cell chloroplasts are smaller and have less granum, hence could be said to be less developed than the mesophyll cells. Guard cell chloroplasts have a reduced thylakoid network and chlorophyll contents compared to the mesophyll [13] and have functional photosystems I and II, electron transport, oxygen evolution, and photophosphorylation [8, 14]. Elucidating the role of the guard cell chloroplasts and guard cell photosynthesis presents a serious challenge for researchers in the field but the following roles have been proposed for guard cell chloroplasts.

1. Electron transport in guard cells is capable of producing ATP used in osmoregulation [15, 16, 17].

2. Photosynthetic carbon assimilation within guard cells produces osmotically active sugars [8, 12, 17, 18].

3. Chloroplasts are involved in blue-light signaling and response [19, 20].

4. Starch stored in the chloroplasts (either produced from carbon assimilation in the guard cell chloroplasts or imported from the mesophyll) is available to synthesize malate as a counter ion to K⁺ or is degraded into sucrose.

1.2 Electron transport in guard cells

Linear electron transport and photophosphorylation in the guard cells have been documented. The quantum efficiency for PSII photochemistry in guard cells has been shown to the rate of 70 to 80% that of mesophyll cells when subjected to a range of light levels thereby suggesting a similarity of operation in mechanisms in both guard and mesophyll cell [14, 21]. The pigment composition of guard cells is also similar to that of the mesophyll cells [22], which leads to enhance ATP production. As a result, such electron transport rate could provide sufficient energy to drive ions for stomatal opening in the absence of CO₂ fixation. Using the high-resolution chlorophyll fluorescence imaging, [14, 23] found that Rubisco was a major sink for the products of electron transport suggesting that guard cell electron transport can be mediated by [CO₂] meaning Calvin cycle activity does take place in the guard cell [24].

1.3 Advances, tools, and techniques for chloroplasts and guard cells manipulation toward plants improvements

Recent developments in technology have opened opportunities to explore guard cell mechanisms, and therefore potentials for regulating WUE and enhancing plant productivity. Efficient, simple, and fast cloning techniques are available for the design of desired single or multiple genes to be expressed in plants for genetic manipulation of metabolites involved in photosynthetic metabolism, which could lead to changes in stomatal behavior and potentially improve photosynthesis and water use efficiency in plants [25].

Specific cell metabolisms in guard and mesophyll cells can now be targeted and the possible coordination between mesophyll metabolites in relation to stomatal functions determined. The development of guard cell-specific promoters has made these manipulations of expression of specific gene transcripts possible, which provides opportunities to manipulate guard cell-specific metabolisms or specific stomatal traits in order to elucidate their functions or interactions. For instance, KST1 and MYB60 promoters, which are guard cell-specific promoters developed by Müller-Röber et al. [26], have been used to drive the expression of target genes specifically in guard cells. The use of a specific guard cell promoter has demonstrated by Wang et al. [27], Kucheli [25] through an enhanced light-induced stomatal opening, greater photosynthesis, and improved growth rate in Arabidopsis overexpressing H⁺-ATPase among others.

Prior studies have targeted both the Calvin cycle and electron transport chain enzymes to expressions in order to identify their control on carbon assimilation [28, 29, 30]. It is now possible to demonstrate single or more enzyme transformations, which have shown to enhance photosynthetic rates in varieties of crops. For instance, increased sedoheptulose-1,7-bisphosphatase activity resulted in tobacco plants with improvements in carbon assimilation by 6–12% [28, 31]. It is in line with this that we elucidated the role of the guard cells chloroplasts using guard cell-specific promoters (Myb60 and KST1) to drive expression specifically in the guard cells in order to produce transgenic plants with manipulation in electron transport chain.

1.4 Design and development of golden gate construct to manipulate expression of cytochrome B₆F (Rieske) in Arabidopsis thaliana

An electron transport chain is a series of complex reactions that transfer electrons from electron donors to electron acceptors alongside the transfer of protons H⁺ ions across a membrane. The cytochrome b₆f complex also known as the plastoquinol-plastocyanin reductase is an enzyme found in the electron transport chain on the thylakoid membrane of chloroplasts. In photosynthesis, the cytochrome b₆f complex catalyzes the transfer of electrons between the two photosynthetic reaction centers, Photosystems II and Photosystem I, while at the same time, transferring protons across the thylakoid used to synthesize ATP from ADP for the Calvin cycle [32, 33].

The overexpression of the Rieske FeS protein in Arabidopsis resulted in substantial improvements of quantum efficiency of PSI and PSII and electron transport, which lead to significant impacts on plant yield [30].

Transgenic approaches have demonstrated striking results of the manipulation of the Calvin cycle where energy conversion led to increasing yield potential [29, 34, 35, 36, 37] suggesting that these studies may imply improvements in photosynthesis through overexpressing the activity of individual enzymes. Already, evidence supporting this hypothesis from single manipulations has been demonstrated from transgenic tobacco plants over-expressing SBPase [28] and also the combined multigene approach of over-expressing SBPase and FBPA [38]. These photosynthetic manipulations resulted in increased carbon assimilation, enhanced growth, and increased cumulative biomass, hence the genetic potentials that lie there. It is, therefore, obvious that number of sense and antisense plants with increased and reduced levels of SBPase and Rieske have varying photosynthetic capacity and have altered carbohydrate status at the whole leaf level, thus leading to modifications in growth and development.

Transgenic plants with guard cells specific manipulation are, therefore, important for identification and characterization of the signal transduction mechanisms [25]. Therefore, guard cell photosynthesis demands genetic analyses by guard cell-specific manipulation of photosynthesis. Multiple targets have been identified and could be manipulated to aid more understanding to maximize crop production. Some of these targets are the SBPase and Rieske enzymes, which have demonstrated of their significance in controlling photosynthetic processes.

2. Materials and methods

2.1 Design and construction of constructs for use in Arabidopsis thaliana in the chloroplasts of guard cells

The sequences of the genes of interest Rieske in Arabidopsis thaliana were retrieved from the TAIR database and primers designed. The plasmid vectors used for the plant transformation were constructed using the golden gate cloning and the Moclo system [39, 40]. Constructs were designed to alter expression of the Rieske genes in a cell-specific manner driven by the KST1 and MYB60 promoters. YFP tags were also included in the construct to demonstrate cell specificity.

2.2 Screening of mutants

Arabidopsis mutants were identified by PCR screening. Arabidopsis stable transformants carrying the transgenes were also screened using antibiotic and/or herbicide. Plants were germinated on agar plates containing antibiotics. For seeds germinated in soil, the soil was treated with 0.82 mM of glufosinate-ammonium and were watered with this until selection was obvious and seedling selected with plants transplanted into individual pots. The presence of the transgene was reconfirmed by genomic DNA PCR screening.

3. Results

3.1 Selection of Arabidopsis transformants

Transformed floral plants in the Arabidopsis plants were allowed to mature to seed. Seeds were collected and planted for the screening of the T1 generation and selection of positive transformants achieved. Selection of positive transformants was identified by the application of BASTA watered on the soil in which the T1 germinated seedlings as transformants were resistant to the herbicide (Figure 2).

Figure 2.

Hebicide (BASTA) selection of transformed Arabidopsis plants. Resistant transformants were selected by growing on soil and spraying with BASTA (presence of bar gene confers resistance to the glofusinate ammonium herbicide BASTA). (a) WT control showing complete death of cotyledons grown on BASTA, (b) WT control grown without BASTA, and (c) selection of resistant transformants grown on soil watered with BASTA. Plants growth conditions maintained under controlled-environment growth room with (22°C, 8 h light, 16 h dark cycle). White scale bar represents 1 cm.

The resulting successful transgenic plants were selected on the herbicide glofusinate ammonium (BASTA) and subsequent screening for homozygous lines began by PCR followed by confirmation by iDNA technology.

4. DNA analysis of transformed plants

The result selected from transformed individual plants was checked for the presence of the transgene by PCR analysis. The result of the DNA analysis produced PCR fragment sizes exactly as the gene of interest in all the lines screened. Constructs have shown all ten lines selected positive for the presence of the transgene (Figure 3).

Figure 3.

Genomic DNA PCR screening of transformants for presence of the transgene. The presence of transgenes was checked by PCR analysis of genomic DNA of plants). Ten lines were screened per construct. WT DNA (WT + red) and plasmid DNA containing the gene of interest (P+) were used as negative and positive controls, respectively. PCR products were run alongside molecular weight marker (DNA generuler ladder mix from thermoscientific) in base pairs.

5. Fluorescence microscopy to detect YFP expression in chloroplasts of guard cells

Rieske YFP mutants were rapidly screened for the presence and localization of the yellow fluorescence protein (YFP) specifically in guard cells using high-resolution chlorophyll fluorescence microscope. Constructs fused to the yellow fluorescence protein confirmed that expressions driven by the cells specific promoters were confined to the chloroplasts in the guard cells. Constructs tagged with either the MYB60 promoter or KST1 promoter revealed the chloroplasts with the YFP in them while wild-type control had no signal (Figure 4).

Figure 4.

Specific YFP expression in the guard cell of transformed plants. Localization of the YFP in the chloroplasts of guard cells of Arabidopsis transformants. (a) Wild-type (Col-0) tissue showing no signal while (b) expression of the constructs tagged with YFP and driven by the MYB60 promoter in leaf tissue was checked using the high-resolution microscope. Images acquired by exiting with 515 nm LEDs and emission collected with a band pass filter 530 ± 20).

6. Photosystem II operating efficiency and growth analysis

The first obvious thing observed in the transgenic lines was the clear evidence of phenotypes in all the constructs selected for further analysis.

Observations revealed developmental phenotypes in the early stage of plants growth between the WT and transgenic lines. The total rosettes or leaf area of the transgenic plant of the lines within the construct 4-pL2B-BAR-(pKST1)-AtRieske-tHSP evidently showed larger leaf area (Figure 5).

Figure 5.

Growth phenotype of WT and homozygous mutant lines of construct 4- (pL2B-BAR-(pKST1)-AtRieske-tHSP plants grown on soil. 4–weeks old plants were germinated and grown for 14 days on soil before picked out and transferred individually to pots. Plants were grown under identical conditions in a controlled-environment growth room with (22°C, 8 h light, 16 h dark cycle). Arabidopsis thaliana (Col-0) WT and mutant lines are shown. White scale bar represents 5 cm.

Chlorophyll fluorescence imaging was also performed on the plants and the operating efficiecny of PSII photochemistry (Fq′/Fm′) determined. Chlorophyll fluorescence imaging of WT and mutant lines of construct 4-(pL2B-BAR-(pKST1)-AtRieske-tHSP to changes in light intensity subjected to 150 and 600 μmol m ⁻² s⁻¹ showed significant differences between the wild type and mutants in the first 3 and 4 weeks of recording confirming also the phenotype found in the images observed above. However, as the plants advanced in the next 2–3 weeks, the reductions in Fq'/Fm′ of the wild type seemed to catch up with the transgenic lines in all constructs suggesting that the expression of these genes might be most critical in their early stages of development. There were no significant differences found in the later weeks of the experiment between the wild type and the transgenics (Figure 6).

Figure 6.

Chlorophyll fluorescence imaging comparison of WT and mutant lines of construct 4-(pL2B-BAR-(pKST1)-AtRieske-tHSP to changes in light intensity. The maximum PSII operating efficiency (Fq′/Fm′) values of the whole plant subjected to (a)150 μmol m -2 s-1 and (b) 600 μmol m -2 s-1 were measured. Plants were germinated and grown for 14 days on soil before picked out and transferred to individual pot on soil. These were grown for additional 5 weeks under identical conditions in a controlled environment growth room (22°C, 8 h light, 16 h dark cycle). Arabidopsis thaliana (Col-0) WT and mutants individual lines of the construct 4-(pL2B-BAR-(pKST1)-AtRieske-tHSP measured. Data were obtained using 10–15 individual plants from three independent transgenic lines and are derived from weeks 3, 4, 5, and 7. Columns represent mean values, and standard errors are displayed, respectively. Significant differences between WT and lines (P < 0.05) at weeks 3 and 4 at 150 μmol m -2 s-. Each line was significantly different from wild-type WT. At higher light level of 600 μmol m -2 s-.1 no difference between the wild type and transgenic found in all the lines.

Chlorophyll fluorescence imaging used to determine the maximum PSII operating efficiency of photosynthesis (Fq′/Fm′) values of the whole plant was also used to capture images at the time of analysis. These images further tell more of the PSII efficiency differences indicated by colors from the scale, which ranges from green (lowest value) to orange (highest value). The more the value, the more efficient is the PSII efficiency. Images likewise show the growth differences, which were evident between the wild type and transgenic lines. The PSll photosynthetic differences of all the four constructs at week 3 are shown in the Figure 7 below, which clearly showed a faster growth phenotype with larger rosettes in the transgenic lines than the wild type.

Figure 7.

Photosynthetic efficiency of PSll operating systems of images captured of WT and homozygous mutant Arabidopsis plants. 3–weeks old plants were germinated and grown for 14 days on soil before picked out and transferred to individual pots on soil. Plants were grown under identical conditions in a controlled-environment growth room with (22°C, 8 h light, 16 h dark cycle). Arabidopsis thaliana (Col-0) WT and mutants are shown. Scale bars represent 5 cm and Fq′/Fm′ values represented by colors as indicated.

Growth was slower in the wild type in the early stages as seen above. However, as the plants advanced (week 7), the reductions in rosette area of the wild type seemed to catch up with the transgenic lines, which might again be implying that the expression of these two genes might be more active in early developmental stages.

7. Discussion

Transgenic studies have provided numerous evidence that manipulation of certain enzymes is potential route for the improvement of plant productivity [14, 28, 31, 36, 38, 41, 42].

In this work, we generated transgenic Arabidopsis (Arabidopsis thaliana) plants overexpressing the Rieske enzyme mutant lines with altered manipulation identified in Arabidopsis thaliana. The analysis carried out such as the DNA and herbicides screening plus the localization of the YFP have all revealed that these genes are present in the chloroplasts of the guard cells and had impact on the transgenic lines.

Phenotypes observed suggested that the transformed plants exhibited significant faster initial growth rates evidenced by larger leaf area and faster rosette increases which may suggest that Rieske might be of importance for enhanced plant growth. Studies have reported of increased yield in plant productivity by overexpressing these genes in the whole plants [28, 30, 36, 41]; however, it is interesting that we have observed similar enhancement of growth despite expression being limited to guard cells. The quantum efficiency of PSII photochemistry in the transgenic lines was also significantly higher in early development. These data imply that the photosynthetic efficiency of young plants may have a greater impact on plant development. These findings are consistent with earlier studies, which reported that the stimulatory effects of increased levels of SBPase occurred earlier in development [28], which may also demonstrate the different limitations witnessed on photosynthesis between developing and fully expanded leaves.

It is important also to keep in mind that little changes in photosynthetic capacity counts and can have a great impact on plant development [28]. Therefore, these results suggest that altered expression (assumed due to the expression of the construct) Reiske in guard cells alone in plants seem to improve the overall plant photosynthetic efficiency and growth in these plants compared with the wild type suggesting that genes manipulation in the guard cells may be playing roles in plant development.

In conclusion, numerous studies have shown that photosynthetic enzymes in carbon metabolism have yielded increased photosynthetic rates in plant at the whole leaf level [34, 36, 41, 42, 43, 44, 45].

This research was based on elucidating the role of guard cells chloroplasts in stomatal regulation, and the role these cells could play in enhancing plant productivity. A comprehensive understanding of the signals or metabolites synchronizing stomatal conductance and carbon assimilation is, therefore, paramount toward successful manipulations of stomatal behavior for enhancing water use efficiency and sustainable inputs in agriculture.

The molecular approach efforts to comprehend the involvement of guard cell photosynthesis in stomatal function required manipulating photosynthesis specifically in guard cells, which demonstrated the potential of transgenic plants with altered guard cell metabolism. We particularly have demonstrated specificity of the KST promotor and shown that expression was only in the guard cells. This has also shown the potential of this promotor for manipulating guard cell-specific metabolism.

8. Additional information

Parts of this chapter were previously published in a doctoral thesis by the same author titled “The role of guard cell chloroplasts in stomatal function and coordinating stomatal and mesophyll responses” at the University of Essex, UK 2018.