Guard-Cell-Specific Expression of Phototropin2 C-Terminal Fragment Enhances Leaf Transpiration

Phototropins (phot1 and phot2) are plant-specific blue light receptors that mediate chloroplast movement, stomatal opening, and phototropism. Phototropin is composed of the N-terminus LOV1 and LOV2 domains and the C-terminus Ser/Thr kinase domain. In previous studies, 35-P2CG transgenic plants expressing the phot2 C-terminal fragment–GFP fusion protein (P2CG) under the control of 35S promoter showed constitutive phot2 responses, including chloroplast avoidance response, stomatal opening, and reduced hypocotyl phototropism regardless of blue light, and some detrimental growth phenotypes. In this study, to exclude the detrimental growth phenotypes caused by the ectopic expression of P2C and to improve leaf transpiration, we used the PHOT2 promoter for the endogenous expression of GFP-fused P2C (GP2C) (P2-GP2C) and the BLUS1 promoter for the guard-cell-specific expression of GP2C (B1-GP2C), respectively. In P2-GP2C plants, GP2C expression induced constitutive phototropin responses and a relatively dwarf phenotype as in 35-P2CG plants. In contrast, B1-GP2C plants showed the guard-cell-specific P2C expression that induced constitutive stomatal opening with normal phototropism, chloroplast movement, and growth phenotype. Interestingly, leaf transpiration was significantly improved in B1-GP2C plants compared to that in P2-GP2C plants and WT. Taken together, this transgenic approach could be applied to improve leaf transpiration in indoor plants.

Identification of DIR1-Dependant Cellular Responses in Guard Cell Systemic Acquired Resistance

After localized invasion by bacterial pathogens, systemic acquired resistance (SAR) is induced in uninfected plant tissues, resulting in enhanced defense against a broad range of pathogens. Although SAR requires mobilization of signaling molecules via the plant vasculature, the specific molecular mechanisms remain elusive. The lipid transfer protein defective in induced resistance 1 (DIR1) was identified in Arabidopsis thaliana by screening for mutants that were defective in SAR. Here, we demonstrate that stomatal response to pathogens is altered in systemic leaves by SAR, and this guard cell SAR defense requires DIR1. Using a multi-omics approach, we have determined potential SAR signaling mechanisms specific for guard cells in systemic leaves by profiling metabolite, lipid, and protein differences between guard cells in the wild type and dir1-1 mutant during SAR. We identified two long-chain 18 C and 22 C fatty acids and two 16 C wax esters as putative SAR-related molecules dependent on DIR1. Proteins and metabolites related to amino acid biosynthesis and response to stimulus were also changed in guard cells of dir1-1 compared to the wild type. Identification of guard cell-specific SAR-related molecules may lead to new avenues of genetic modification/molecular breeding for disease-resistant plants.

Structural and Functional Insights into the Role of Guard Cell Ion Channels in Abiotic Stress-Induced Stomatal Closure

A stomatal pore is formed by a pair of specialized guard cells and serves as a major gateway for water transpiration and atmospheric CO2 influx for photosynthesis in plants. These pores must be tightly controlled, as inadequate CO2 intake and excessive water loss are devastating for plants. When the plants are exposed to extreme weather conditions such as high CO2 levels, O3, low air humidity, and drought, the turgor pressure of the guard cells exhibits an appropriate response against these stresses, which leads to stomatal closure. This phenomenon involves a complex network of ion channels and their regulation. It is well-established that the turgor pressure of guard cells is regulated by ions transportation across the membrane, such as anions and potassium ions. In this review, the guard cell ion channels are discussed, highlighting the structure and functions of key ion channels; the SLAC1 anion channel and KAT1 potassium channel, and their regulatory components, emphasizing their significance in guard cell response to various stimuli.

Raf-like kinases and receptor-like (pseudo)kinase GHR1 are required for stomatal vapor pressure difference response

Stomatal pores close rapidly in response to low-air-humidity-induced leaf-to-air vapor pressure difference (VPD) increases, thereby reducing excessive water loss. The hydroactive signal-transduction mechanisms mediating high VPD–induced stomatal closure remain largely unknown. The kinetics of stomatal high-VPD responses were investigated by using time-resolved gas-exchange analyses of higher-order mutants in guard-cell signal-transduction branches. We show that the slow-type anion channel SLAC1 plays a relatively more substantial role than the rapid-type anion channel ALMT12/QUAC1 in stomatal VPD signaling. VPD-induced stomatal closure is not affected in mpk12/mpk4GC double mutants that completely disrupt stomatal CO2 signaling, indicating that VPD signaling is independent of the early CO2 signal-transduction pathway. Calcium imaging shows that osmotic stress causes cytoplasmic Ca2+ transients in guard cells. Nevertheless, osca1-2/1.3/2.2/2.3/3.1 Ca2+-permeable channel quintuple, osca1.3/1.7-channel double, cngc5/6-channel double, cngc20-channel single, cngc19/20crispr-channel double, glr3.2/3.3-channel double, cpk-kinase quintuple, cbl1/4/5/8/9 quintuple, and cbl2/3rf double mutants showed wild-type-like stomatal VPD responses. A B3-family Raf-like mitogen-activated protein (MAP)-kinase kinase kinase, M3Kδ5/RAF6, activates the OST1/SnRK2.6 kinase in plant cells. Interestingly, B3 Raf-kinase m3kδ5 and m3kδ1/δ5/δ6/δ7 (raf3/6/5/4) quadruple mutants, but not a 14-gene raf-kinase mutant including osmotic stress-linked B4-family Raf-kinases, exhibited slowed high-VPD responses, suggesting that B3-family Raf-kinases play an important role in stomatal VPD signaling. Moreover, high VPD–induced stomatal closure was impaired in receptor-like pseudokinase GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1) mutant alleles. Notably, the classical transient “wrong-way” VPD response was absent in ghr1 mutant alleles. These findings reveal genes and signaling mechanisms in the elusive high VPD–induced stomatal closing response pathway.

A NET4-RabG3 couple mediate the link between actin and the tonoplast and is essential for normal actin cytoskeletal remodelling in stomatal closure to flg22

Members of the NETWORKED (NET) family are involved in actin-membrane interactions. They tether the cell's plasma membrane (PM) to the actin network. Moreover, in a similar manner, they are also involved in the tethering of membrane bound organelles to the actin cytoskeleton; the endoplasmic reticulum (ER) and the ER to the PM. This raises the question as to whether NET proteins are involved in actin cytoskeletal remodelling. Here we show that two members of the NET family, NET4A and NET4B, are essential for normal guard cell actin reorganization, which is a process critical for stomatal closure in plant immunity. NET4 proteins interact with F-actin and with members of the Rab7 GTPase RABG3 family through two distinct domains, allowing for simultaneous localization to actin filaments and the tonoplast. NET4 proteins interact with GTP-bound, active RABG3 members, suggesting their function as downstream effectors. We also show that RABG3b is critical for stomatal closure induced by microbial patterns. Taken together, we conclude that the actin cytoskeletal remodelling during stomatal closure depends on a molecular link between actin filaments and the tonoplast, which is mediated by the NET4-RABG3b interaction. We propose that stomatal closure to microbial patterns involves the coordinated action of immune signalling events and proper actin cytoskeletal remodelling.

Stomatal responses at different vegetative stages of selected maize varieties of Bangladesh under water deficit condition

Drought stress causes stomatal behavior change in most plants. Water deficit condition caused by drought is one of the most significant abiotic factors reducing plant growth, development, reproductive efficiency, and photosynthesis, resulting in yield loss. Maize (Zea mays L.) holds a superior position among all the cereals due to its versatile use in the food, feed, and alcohol industries. A common demonstrative feature of a complex network of signaling pathways led by predominantly abscisic acid under drought conditions is stomatal aperture reduction or stomatal closure, which allows the plant to reduce water loss through the stomatal pore and to sustain a long time on water deficit condition. This study analyses the stomatal density, stomatal closure percentages, and guard cell aperture reduction using a microscopy-based rapid & simple method to compare guard cell response & morphological variations of three hybrid maize varieties viz. BHM (BARI hybrid maize)-7, BHM-9, and BHM-13 developed by Bangladesh Agricultural Research Institute (BARI). A drought treatment was applied to all varieties at two different vegetative stages, vegetative stage 3 (V3) and V5, until they reach V4 and V6, respectively. After drought exposure at the V4 stage, the percentage of closed stomata of BHM-7, BHM-9, and BHM-13 was 21%, 23%, and 33%, respectively. The reduction in the guard cell aperture ratio of BHM-7, BHM-9, and BHM-13 was 14.83%, 10.92%, and 33.85%, respectively. At the V6 stage, for the second set of plants, the closed stomata of BHM-7, BHM-9, and BHM-13 were 18%, 21%, and 34%, respectively. The rate of reduction in guard cell aperture ratio of BHM-7, BHM-9, and BHM-13 was 5.52%, 2.48%, and 38.75%, respectively. Therefore, BHM-13 showed maximum drought adaptation capacity compared to BHM-7 and BHM-9 due to the highest percentage of closed stomata and the highest percentage of reduction in aperture ratio.

Construction of Sedoheptulose-1,7-Bisphosphatase (Sbpase) for Manipulation in Guard Cells of Arabidopsis thaliana L.

Guard cells control the stomata through which exchange of gas takes place in plants by balancing between CO2 uptake for photosynthesis and water loss through transpiration leading to ultimate plant water use efficiency (WUE). Due to climate change, sustainable agriculture will therefore require a major reduction in plant water use hence stomata have become potential target for manipulation. Understanding the signal mechanisms of stomata is therefore critically important in facilitating an understanding of stomatal regulation. The use of molecular tools and techniques to manipulate chloroplast metabolism specifically in the guard cells are needed to elucidate signals associated with stomatal behaviour towards crop improvement. Ability to assemble multiple or complex DNA molecules containing large number of genetic elements is an essential part of genetic engineering and in order to understand the involvement of guard cell photosynthesis in stomatal function, genetic manipulation of photosynthetic enzymes specifically in guard cells is necessary. This study employed the manipulation and construction of the enzyme Sedoheptulose-1,7-Bisphosphatase (SBpase) by using the golden gate cloning technique and the bioinformatics system- geneious. Constructs were designed to alter expression of the SBPase gene in a cell specific manner driven by the guard cell promoter KST1 in the model plant Arabidopsis thaliana L. The construct design for the sense plasmid vectors allowed efficient assembly of multiple DNA fragments in a single reaction based on the type IIs restriction enzyme. The potentials of manipulating guard cell specific metabolism are therefore enormous and the increase or decrease of photosynthetic genes could be assessed and their impacts on plant development documented accordingly.