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Scientific article on control of stomata

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Scientific article on control of stomata

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Introduction

Stomata are specialized epidermal structures that are essential for plant survival and productivity. These structures consist of two guard cells around a pore. Every stoma is a molecular valve that acts in gas exchange, mainly CO ₂ and O ₂, which is necessary for optimal photosynthesis and which restricts water loss by modulating the transpiration level. The genes that are involved in the process of stomata development were crucial for the movement of plants from water to land during evolution since stomata facilitated gas exchange while limiting desiccation. The stomatal morphogenesis pathway has been identified in detail in Arabidopsis thaliana through investigations of many mutants with an impaired stomatal pattern or with other morphological defects in their epidermal cells. Cell distribution and differentiation require a balance between proliferation and cell specification in time and space. The differentiation of stomata is preceded by at least one asymmetric as well as a few symmetric cell divisions. It requires three different types of precursor cells: the meristemoid mother cell (MMC), meristemoids and the guard mother cell (GMC). The last step of stomatal development is the differentiation of the stoma itself within the structure of the guard cells. The number and pattern of stomata varies in different organs in A. thaliana. A common feature of patterning is that stomata are separated from each other by at least one epidermal cell. This pattern ensures the presence of neighbor cells for ion exchange, which is necessary for the regulation of the aperture width. For this reason, neighbor cells are part of a stomatal complex (Nadeau and Sack, 2002; Nadeau, 2009; Lau and Bergmann, 2012; Pillitteri and Torii, 2012; Vatén and Bergmann, 2012). Recent research has shown that the mode of action of stomata depends on the integration of environmental and intracellular signals. Many environmental factors such as CO ₂ concentration, biotic and abiotic stresses, and additionally different plant hormones, can modulate stomatal reaction. For plants that encounter dehydration stress, the most essential factor is the ability of stomata to close and thus prevent excess water loss. Opening and closing is achieved by the swelling and shrinking of the guard cells, which is driven by ion exchange; cytoskeleton reorganization and metabolite production; the modulation of gene expression and the posttranslational modification of proteins (reviewed in Kim et al., 2010). Swelling of the guard cells results in stomata opening since the content of ions and osmolites within them makes them bigger and thus able to move away from each other making the stomatal aperture larger. In contrast, closing is an opposite mechanism and results in the shrinking of the guard cells when the efflux of ions occurs.

Stomatal closure is the earliest plant response to water deficit (Schroeder et al., 2001b). This rapid reaction is regulated by a complex network of signaling pathways, in which the major and the best-known player, abscisic acid (ABA), acts in concert with jasmonates (JA), ethylene, auxins, and cytokinins (Nemhauser et al., 2006; Huang et al., 2008). The complexity of the response is mainly dependent on the initial threshold of stress and individual plant's stress history. Generally, ABA and JA are positive regulators of stomatal closure, while auxin and cytokinins are positive regulators of stomatal opening. The mode of action of ethylene is ambiguous because it can act as a positive or negative regulator, depending on the tissue and conditions (Nemhauser et al., 2006; Huang et al., 2008).

This paper presents a comprehensive review of the genetic and molecular basis of stomata action under the control of phytohormones, particularly when response to drought stress is considered.

Open or Close the Gate - The Role of ABA, Ion Channels, and Diurnal Cycle in Stomatal Movements Regulation

The regulatory role of ion channels localized in the guard cell membrane in the opening and closing stomata

The guard cell turgor is dynamically adjusted to environmental conditions and hormonal signals in order to facilitate the proper gas exchange and prevent excessive water loss. Mature guard cells do not have plasmodesmata and for this reason most influx and efflux of solutes occurs via ion channels, transporters, and pumps that are localized in the plasma membrane (PM). The action of ion channels, transporters, and pumps that are essential for stomatal function is well documented and supported by molecular studies involving mutants in the genes encoding these protein. During the opening of the stomata, the H⁺-ATPase pump mediates the efflux of H⁺ from the guard cells. In plants, H⁺-ATPases belong to the multi-gene family of the P-type ATPases, with 11 genes in Arabidopsis, which are all expressed in the guard cells (Ueno et al., 2005). In the guard cells, the action of H⁺-ATPase activity is positively regulated by blue light and auxins, whereas Ca ²⁺ and ABA act as negative regulators. The efflux of H⁺ hyperpolarizes the PM and leads to K⁺ uptake via activation of inward K⁺ rectifying channels, such as KAT1 (potassium channel in Arabidopsis thaliana 1), KAT2 (potassium channel in Arabidopsis thaliana 2), and AKT1 ( Arabidopsis thaliana K⁺ transporter 1) (Schachtman et al., 1992; Pilot et al., 2001; Szyroki et al., 2001). Another signal that activates the influx of K⁺ via K⁺ channels is the acidification of the apoplast as a result of H⁺ extrusion from the guard cells. K⁺ uptake is balanced by counter-ions, mainly Cl − obtained from the apoplast, malate 2− that is derived from starch breakdown or . The last one is transported from the apoplast by a nitrate transporter AtNRT1.1 (CHL1) [nitrate transporter 1 (chlorina1)]. The importance of uptake was confirmed by an analysis of an Arabidopsis clh1 mutant. The stomatal apertures of the chl1 mutant were smaller than those of the wild-type when nitrate was supplied. Furthermore, the chl1 mutant was drought tolerant (Guo et al., 2003). Ions supplied into the guard cells together with water transported via aquaporins generate the turgor that are necessary to keep stomata open (Figure A).

During stomatal closure, the inhibition of H⁺-ATPase and the activation of anion channels together result in membrane depolarization. Anion channels such as rapid channels (R-type) and slow channels (S-type) facilitate the efflux of malate 2−, Cl −, and (Roelfsema et al., 2004; Roelfsema and Hedrich, 2005). The decreased level of malate 2− in guard cells is also linked with the gluconeogenic conversion of malate 2− into starch (Willmer and Fricker, 1996). Membrane depolarization creates a driving force for the efflux of K⁺ via K⁺ outwardly rectifying channels such as GORK (guard cell outwardly rectifying K⁺ channel) (Jeanguenin et al., 2008). An Arabidopsis gork mutant displayed impaired stomatal closure, thus confirming the important role of GORK in elimination K⁺ ions and in the facilitation of stomatal closure (Hosy et al., 2003). Another event that accompanies stomatal closure is an elevation of the cytoplasmic Ca ²⁺ concentration as a result of Ca ²⁺-release via channels situated in both the PM and in the tonoplast (MacRobbie, 2006). Ca ²⁺ channels are encoded by genes from three gene-families: TPC1 (two-pore channel 1) (Peiter et al., 2005), CNGC (cyclic nucleotide gated channel) (Finn et al., 1996), and GLR (glutamate receptor) (Lacombe et al., 2001). Taken together, the efflux of solutes from the guard cells leads to a reduced turgor and stomatal closure (Figure B).

Abscisic acid - how the proper level of the main regulator of stomatal movements is achieved in plants

Abscisic acid has been postulated as a main regulator of stomatal movements but its proper functioning depends on the appropriate level of biologically active ABA within the plant cells. This is achieved by synchronized processes such as ABA biosynthesis, catabolism, conjugation/deconjugation, and transport. These processes, which are well recognized and studied in various species, have confirmed the function of many enzymes involved in the biosynthesis, catabolism, conjugation/deconjugation, and transport of ABA. The exception, not fully recognized yet, is ABA signal transduction pathway. Although ABA has been the focus of many research groups since the early 90s, there are still many questions in regards to the function of the proteins involved in ABA signaling, protein interactions or the impact of the components of signalosome on specific physiological responses. Therefore, with the progress in studies on ABA signaling, the state of knowledge and the already known interaction web should be updated and verified.

Abscisic acid is synthesized in the plastids and cytosol, mainly in the vascular parenchyma cells but also in the guard cells, through the cleavage of a C40 carotenoid precursor, followed by a two-step conversion of the intermediate xanthoxin into ABA via ABA-aldehyde (Taylor et al., 2000; Finkelstein and Rock, 2002; Schwartz et al., 2003; Endo et al., 2008; Melhorn et al., 2008). The pathway begins with isopentenyl pyrophosphate (IPP), which is the biological isoprene unit and the precursor of all terpenoids, as well as many plant hormones. The next step is the epoxidation of zeaxanthin and antheraxanthin into violaxanthin, which is then catalyzed by zeaxanthin epoxidase (ZEP) (Marin et al., 1996). After a series of violaxanthin modifications that are controlled by the enzyme ABA4, violaxanthin is converted into 9-cis-epoxycarotenoid (North et al., 2007). Oxidative cleavage of the major epoxycarotenoid 9-cis-neoxanthin by the 9-cis-epoxycarotenoid dioxygenase (NCED) yields a C15 intermediate - xanthoxin (Schwartz et al., 1997). This step is the last one that occurs in plastids. Xanthoxin is exported to the cytoplasm where a two-step reaction via ABA-aldehyde occurs. The first step is catalyzed by a short-chain alcohol dehydrogenase/reductase (SDR) that is encoded by the AtABA2 ( ABA deficient 2) gene (Rook et al., 2001; Cheng et al., 2002; Gonzalez-Guzman et al., 2002) and that generates ABA-aldehyde. Then, the ABA-aldehyde oxidase (AAO) with the molybdenum cofactor (MoCo) catalyzes the last step in the biosynthesis pathway - the conversion of ABA-aldehyde into ABA (Seo et al., 2004) (Figure A). The appropriate level of active ABA is achieved not only through the biosynthesis and catabolism reactions performed by CYP707A1-4 (cytochrome P450, family 707, subfamily A, polypeptide 1, 2, 3, 4) (Kushiro et al., 2004; Figure B), but also by the inactivation of ABA through conjugation and deconjugation. ABA can be inactivated at the C-1 hydroxyl group by different chemical compounds that form various conjugates and that accumulate in vacuoles or in the apoplastic space (Dietz et al., 2000). The most widespread conjugate is ABA glucosyl ester (ABA-GE), which is catalyzed by ABA glucosyltransferase (Boyer and Zeevaart, 1982). Lee et al. ( 2006) identified the AtBG1 (beta-1,3-glucanase 1) protein that is responsible for the release of ABA from ABA-GE. Their findings showed that ABA deconjugation plays a significant role in providing an ABA pool that allows plants to adjust to changing physiological and environmental conditions (Figure C).

The ability of ABA to move long distances allows it to serve as a critical stress messenger. Kuromori et al. ( 2011) identified the ABA importer - ABCG22 ( Arabidopsis thaliana ATP-binding cassette G22). The gene encoding this transporter is mainly expressed in the guard cells. In addition, the expulsion of ABA into the intercellular space is mediated by transporters such as ABCG25 ( Arabidopsis thaliana ATP-binding cassette G25). ABCG25 is expressed primarily in vascular tissues where ABA is synthesized (Kuromori et al., 2010). ABA delivery to the guard cells promotes a cascade of reactions that lead to stomatal closure and that inhibit stomatal opening in order to prevent water loss (Figure D).

After ABA is received from ABC transporters by the guard cells, the PYR/PYL/RCAR (pyrabactin-resistance 1/pyrabactin-resistance like/regulatory component of ABA receptor) perceives ABA intracellularly and forms complexes that inhibit clade A of PP2Cs (protein phosphatase 2C), the negative regulators of ABA signaling, such as ABI1 (ABA insensitive 1), ABI2 (ABA insensitive 2), HAB1 (hypersensitive to ABA1) (Ma et al., 2009; Park et al., 2009; Santiago et al., 2009; Nishimura et al., 2010). The inactivation of PP2Cs allows downstream targets to be phosphorylated and activated - Sucrose Non-fermenting 1-Related subfamily 2 protein Kinases (SnRK2) (Fujii and Zhu, 2009; Fujita et al., 2009; Umezawa et al., 2009; Kim et al., 2010). ABA receptors, PP2Cs, and SnRKs form the core of the early ABA signaling cascade (Figure E).

Regulation of stomatal movements during the diurnal cycle - the role of ABA

The ABA mode of action is linked to diurnal stomatal movements. It has been proposed that this link is based on both the molecular connections between ABA and circadian-clock pathways and on ABA biosynthesis and response to light (reviewed in Tallman, 2004). Although several studies have been carried out linking the diurnal cycle with ABA signaling, there is still a need for further research that would clarify this connection. It has been confirmed that the elevated ABA levels in the dark phase of the day are responsible for stomatal closure but, on the other hand, the molecular basis of the sensing CO ₂ molecules by guard cells is still not well understood. This part of investigations still needs confirmation through the use of well-established methods.

In darkness, stomata are closed. This is probably caused by an intensive ABA accumulation through the biosynthesis of ABA in the guard cells and the simultaneous import of endogenous ABA from the apoplast to the guard cells using ABA transporters such as ABCG22 (Kuromori et al., 2011), while at the same time, ABA catabolism processes are disfavored. Elevated ABA levels cause stomata closure via the activation of an ABA signaling cascade, the efflux of Ca ²⁺ from internal stores, the activation of S-type and R-type anion channels that lead to the efflux of Cl −, malate 2−, and and the activation of the GORK channel that leads to the efflux of K⁺. During the night, elevated levels of CO ₂ in the leaves were observed due to respiration. It has been proved that CO ₂ has a positive effect on the stomatal closure process. The guard cells probably do not sense CO ₂ molecules but instead is synthesized from CO ₂ (Hu et al., 2010), which activates S-type channels and leads to the efflux of Cl −, malate 2−, and (Xue et al., 2011) (Figure A).

At first light, a depletion of endogenous ABA is observed through xanthophyll cycling, the isomerization of ABA precursors and the activation of ABA catabolism enzymes, such as CYP450 (cytochrome P450). The degradation of ABA liberates the guard cells to extrude H⁺ via H⁺-ATPase (H⁺-pump) and accumulate water and ions, such as K⁺, Cl −, malate 2− in order to generate the turgor needed to keep stomata open. K⁺ uptake is mainly responsible for the rapid increase of the turgor and the opening of stomata during the dawn (Humble and Raschke, 1971; Talbott and Zeiger, 1996). The accumulation of sugars such as glucose, fructose and sucrose has been reported during the light phase of the day (Talbott and Zeiger, 1998). In the midday, ABA is delivered to the apoplast around the guard cells through the xylem transpiration stream and the guard cells are regulated by steady-state ABA concentrations (Figure B).

In the evening, ABA biosynthesis outweighs the ABA catabolism in the guard cells, which leads to stomatal closure (for review, see Tallman, 2004).

ABA on the way to reaching the guard cells under drought stress conditions

Under drought stress conditions, ABA would reach a concentration high enough to cause ion efflux and an inhibition of sugar uptake by the guard cells in the midday, thus reducing the apertures for the rest of the day. Analyses of ABA biosynthesis, catabolism, de/conjugation, and transport have been supported by various studies involving several species and different methods, such as mutant analysis, transcriptomics, proteomics, or immunohistochemical techniques. In order to define the role of ABA in stress response, the action of several components of the pathways mentioned were tested in response to stress. The engagement of such various techniques makes the state of knowledge in the field of ABA biosynthesis, catabolism, de/conjugation, and transport well supported and reliable.

It has been shown that ABA concentrations can increase up to 30-fold in response to drought stress (Outlaw, 2003). Water deficit promotes ABA biosynthesis via the upregulation of a key enzyme - NCED3. A significant increase in NCED transcript levels can be detected within 15-30 min after leaf detachment or dehydration treatment (Qin and Zeevaart, 1999; Thompson et al., 2000), which indicates that the activation of NCED genes can be fairly quick. Cheng et al. ( 2002) reported that the AtNCED3, AtZEP ( Zeaxanthin epoxidase), and AtAAO3 ( ABA-aldehyde oxidase) genes could be induced in Arabidopsis by ABA and studies in rice showed that OsNCED3 expression was induced by dehydration (Ye et al., 2011). An immunohistochemical analysis, using antibodies raised against AtNCED3, revealed that protein is accumulated in the leaf vascular parenchyma cells in response to drought stress. This was not detected in non-stressed conditions. These data indicate that drought-induced ABA biosynthesis occurs primarily in the vascular parenchyma cells and that vascular-derived ABA might trigger stomatal closure via the transport to the guard cells (Endo et al., 2008). AtNCED3 expression is upregulated by drought conditions across the species observed and decreases after rehydration.

Drought, like the dark part of a diurnal cycle, also promotes the deconjugation of the ABA-glucose ester (ABA-GE), which is stored in the vacuoles of leaf cells and also circulates in the plant (Xu et al., 2002; Seiler et al., 2011). Both processes, intensive ABA biosynthesis and ABA deconjugation, lead to the accumulation of high levels of biologically active ABA. ABA delivery to the guard cells via ABCG transporters, such as AGCG22 that was mentioned above, promotes a cascade of reactions that lead to stomatal closure and that inhibit stomatal opening in order to prevent water loss (Figure ).

ABA triggers changes in ion homeostasis in the guard cells, which leads to stomatal closure under stress

The ABA signaling network that leads to stomatal closure under stress is activated by the perception ABA. This begins a cascade of reactions that leads to the reduced turgor of the guard cells through ABA modulation of ion channel activities, including the regulated efflux of anions and potassium ions and the inhibition of K⁺ import. Recently, the core signalosome of ABA signaling including ABA receptors, phosphatases (PP2Cs), and kinases (SnRK2s) was established (Ma et al., 2009; Park et al., 2009; Santiago et al., 2009; Nishimura et al., 2010). Although its function is clear and confirmed by advanced molecular analysis, there is still a need to explain the impact of single components, such as kinases, on the regulation of the ion channels or the proton pump (e.g., AHA1), which is described below. On the other hand, the interaction between ABA regulated kinases SnRK2s and S-type anion channels, and the potassium inwardly rectifying channels, described below, has been well established and documented.

The inactivation of PP2Cs such as ABI1 and ABI2 by the complex ABA-receptor facilitates the phosphorylation and activation of a downstream target of phosphatases - SnRK2, such as SnRK2.2/D, SnRK2.3/E, and SnRK2.6/OST1/E, which are the key players in the regulation of ABA signaling and abiotic stress response (Fujii and Zhu, 2009; Fujita et al., 2009; Umezawa et al., 2009). Kinases are able to regulate the activity of ion channels and the proton pump. It was shown that ABA inhibits the action of a proton pump such as H⁺-ATPase. The dominant Arabidopsis mutant ost2 ( opened stomata 2) in AHA1 (H⁺-ATPase 1 HA1) gene exhibited the constitutive activation of AHA1 H⁺-ATPase, which in turn resulted in an inability to close stomata in response to ABA (Merlot et al., 2007). The molecular mechanism of the inhibition of AHA1 by ABA has not yet been fully elucidated. One of the most direct pieces of evidence of the regulation of H⁺-ATPase by SnRK is the demonstration that specific calcium-stimulated kinase, PSK5 (a member of the SnRK3 kinase family), is able to phosphorylate the closest homolog of AHA1 - AHA2 (H⁺-ATPase 1 HA2) in Ser392 localized in the C-terminus of the AHA2 protein. This reaction prevents the 14-3-3 protein, which is the main activator of AHA2 leading to the inhibition of H+-ATPase action, from binding (Fuglsang et al., 2007).

Sucrose Non-fermenting 1-Related subfamily 2 protein Kinases also regulate S-type anion channels and potassium inwardly rectifying channels such as SLAC1 (slow anion channel-associated 1) and KAT1 (K⁺ channel in Arabidopsis thaliana), respectively. The first one is activated by SnRK2, whereas KAT1 is inhibited. SLAC1 encodes the anion-conducting subunit of an S-type anion channel. In different species, S-type anion channels are activated in the guard cells by ABA, cytosolic Ca ²⁺, and phosphorylation events (Schmidt et al., 1995; Pei et al., 1997; Leonhardt et al., 1999; Raschke et al., 2003; Roelfsema et al., 2004; Mori et al., 2006). The slac1 mutant displayed a strongly impaired response to a range of stomatal closing stimuli such as ABA and Ca ²⁺ (Negi et al., 2008; Vahisalu et al., 2008). Increased SLAC1 activity causes an efflux of anions which results in depolarization of the membrane as a consequence of phosphorylation by SnRK. This in turn leads to the loss of K⁺ cations from the cell through the K⁺ efflux channel GORK (guard cell outward-rectifying K⁺), which is activated by depolarization (Jeanguenin et al., 2008). KAT1 is an inward K⁺ channel that allows an influx of K⁺ inside the guard cell when the proton pump drives the PM to a negative potential. When plants encounter drought stress conditions and the ABA level rises, both the proton pumps (as mentioned above) and KAT1 are inactivated by SnRKs. It was shown that the activity of KAT1 is inhibited by an elevation of ABA and cytosolic Ca ²⁺ (Schroeder and Hagiwara, 1989; Blatt and Armstrong, 1993; Grabov and Blatt, 1999) via phosphorylation by SnRK, which in turn results in a decreased influx of K⁺ into the guard cells (Hubbard et al., 2010). The loss of K⁺ and anions from the guard cells is accompanied by the efflux of water via aquaporins. Together, these events lead to a reduction of the turgor, which results in stomatal closure in response to ABA as a major signal of drought (Figure A).

Abscisic acid activates the Ca ²⁺-permeable channels in the PM of the guard cells and triggers an influx of Ca ²⁺ into the cytoplasm of the guard cells through the release of the second messenger, inositol-1,4,5-triphosphate (IP ₃), which in turn activates the Ca ²⁺ channels that are located in the vacuole and endoplasmic reticulum (Schroeder and Hagiwara, 1990; Hamilton et al., 2000; Krinke et al., 2007; Kwak et al., 2008). Ca ²⁺-dependent protein kinases (CDPKs) are activated during drought stress and are able to control stomatal closure in an ABA-dependent manner. After ABA is perceived by a receptor, the action of PP2Cs such as ABI1 are inhibited. ABI1 was identified as a negative regulator of CPK21 (Ca ²⁺ dependent protein kinase 21), which like SnRK phosphorylates SLAC1. SLAC1 phosphorylation, in turn, results in the activation of anion and the efflux of K⁺ (Geiger et al., 2010). An increased cytosolic Ca ²⁺ level activates the Ca ²⁺-dependent pathways that inhibit K⁺ import and trigger the depolarization of the membrane. Mori et al. ( 2006) identified two calcium-dependent kinases - CPK3 (calcium-dependent protein kinase 3) and CPK6 (calcium-dependent protein kinase 6) as positive regulators of ABA signaling in the guard cells during water stress. Inactivation of both genes led to a reduction in the activation of S-type channels by ABA and Ca ²⁺, the impairment of the ABA activation of Ca ²⁺ permeable channels and a decreased sensitivity of stomata to ABA. Disruption of the regulatory subunit RCN1 ( roots curl in NPA) of the gene encoding PP2A (protein phosphatase 2A) led to a reduction of the ABA activation of anion channels and a decreased sensitivity of stomata to ABA (Kwak et al., 2002, Figure ).

Another protein involved in ABA signaling in stomata is GPA1. GPA1 is a positive regulator in the ABA-mediated inhibition of stomatal opening. Arabidopsis plants lacking GPA1 (Gα subunit of G protein) showed a reduction in the inhibition of inward K⁺ currents and a reduced guard cell ABA-insensitivity in stomatal opening (Wang et al., 2001). The mutants era1 ( enhanced response to ABA1) and abh1 ( ABA hypersensitive 1), which are deficient in a farnesyl transferase subunit and RNA cap-binding protein, respectively, are ABA hypersensitive and showed an enhanced ABA activation of S-type channels (Pei et al., 1998; Schroeder et al., 2001a; Hugouvieux et al., 2002; Figure ). However, the exact molecular role of ERA1 or ABH1 in stomatal regulation should be clarified in future research.

During stomatal closure, slow vacuolar (SV) channels activated by cytosolic Ca ²⁺ contribute to the release of Ca ²⁺ from vacuoles. SV channels were shown to be calcium permeable and it was suggested that they facilitate a brief transient efflux of cations, including Ca ²⁺, from vacuoles (Ward and Schroeder, 1994).

Several of the genes involved in the processes described above and more are presented in Table together with a description of mutant phenotypes.

Table 1

Encodes a nuclear cap-binding protein that forms a heterodimeric complex with CBP20 and is involved in ABA signaling

ABA hypersensitive, shows enhanced ABA activation of S-type channels

Schroeder et al. ( 2001a), Hugouvieux et al. ( 2002)

Encodes an anion transporter involved in stomatal closure

Impaired stomatal closure in response to ABA, darkness and CO ₂

Meyer et al. ( 2010)

Encodes a protein containing Leu-rich repeats and a degenerate F-box motif

Disrupted activation of S-type anion channels

Munemasa et al. ( 2007, 2011)

Encodes the calcium-dependent protein kinase 3 (CPK3), a member of the Arabidopsis CDPK gene family. CPK3 is expressed in both guard cells and mesophyll cells. Functions in guard cell ion channel regulation

Reduction in ABA and Ca ²⁺ activation of S-type channels, impaired ABA activation of Ca ²⁺ permeable channels, decreased ABA sensitivity to stomatal closure

Mori et al. ( 2006)

Encodes the calcium-dependent protein kinase 3 (CPK3), a member of the Arabidopsis CDPK gene family. CPK3 is expressed in both guard cells and mesophyll cells. Functions in guard cell ion channel regulation

Reduction in ABA and Ca ²⁺ activation of S-type channels, impaired ABA activation of Ca ²⁺ permeable channels, decreased ABA sensitivity to stomatal closure

Mori et al. ( 2006), Munemasa et al. ( 2011)

Encodes a member of the ERF (ethylene response factor) subfamily B-1 of the ERF/AP2 transcription factor family (ATERF-7). The protein contains one AP2 domain. Phosphorylated by PKS3 in vitro. Involved in ABA-mediated responses

Increased sensitivity of stomata to ABA compared to the wild-type, enhanced drought tolerance

Song et al. ( 2005)

Encodes an alpha subunit of a heterotrimeric GTP-binding protein. GPA1 is a positive regulator in ABA-mediated inhibition of stomatal opening

Reduction in the inhibition of inward K⁺ currents, reduced guard cell ABA-insensitivity in stomatal opening

Wang et al. ( 2001)

Encodes a high-affinity inositol hexakisphosphate transporter that plays a role in guard cell signaling and phytate storage. It is a member of the MRP subfamily/ABC transporter subfamily C

Impaired ABA regulation of Ca ²⁺ permeable channels, defects in S-type channel regulation

Suh et al. ( 2007)

Encodes a putative transcription factor of the R2R3-MYB gene family. Transcript increases under conditions that promote stomatal opening (white and blue light) and decreases under conditions that trigger stomatal closure (ABA, desiccation, darkness) with the exception of elevated CO ₂. Expressed exclusively in the guard cells of all tissues. It is required for light-induced opening of stomata

Reduced stomatal aperture which helps to limit water loss during a drought

Cominelli et al. ( 2005)

Encodes NRT1.1 (CHL1), a dual-affinity nitrate transporter. The protein is expressed in guard cells and functions in stomatal opening

Lower transpiration rate and tolerant to drought

Guo et al. ( 2003)

Encodes a membrane protein with 10 predicted transmembrane helices. SLAC1 is a multispanning membrane protein that is expressed predominantly in the guard cells that play a role in regulating cellular ion homeostasis and S-type anion currents. SLAC1 is important for normal stomatal closure in response to a variety of signals including elevated CO ₂, ozone, ABA, darkness and humidity. SLAC1:GFP localizes to the plasma membrane

Reduced stomatal closure response to ABA, CO ₂, Ca ²⁺ and ozone treatments

Vahisalu et al. ( 2008)

NO and ROS in response to drought stress and ABA

The guard cells generate reactive oxygen species (ROS) such as hydrogen peroxide (H ₂O ₂) and nitric oxide (NO) in response to ABA (Pei et al., 2000; Zhang et al., 2001). Exogenous H ₂O ₂ activates permeable Ca ²⁺ channels in the PM of Arabidopsis guard cells and inhibits inward K⁺ channels (Zhang et al., 2001; Kohler et al., 2003; Kwak et al., 2003). Taking into account the fact that ROS and NO signaling is not yet fully understood, there is a need for further analysis in order to elucidate their function, for example, the role of Ca ²⁺ in ROS and NO action in guard cells should be clarified.

Reactive oxygen species production in Arabidopsis guard cells is mediated by two subunits of NADPH oxidase - AtrbohD ( Arabidopsis thaliana respiratory burst oxidase homolog D) and AtrbohF ( Arabidopsis thaliana respiratory burst oxidase homolog F). The significance of ROS involvement in stomatal closure was revealed by an analysis of the atrbohD/ atrbohF double mutant, which showed impaired stomatal closure in response to ABA (Kwak et al., 2003). The protein, OST1 (open stomata1), displays dominant kinase activity during drought stress response and is able to activate NADPH oxidase (Sirichandra et al., 2009). Mutants in OST1 showed a wilty phenotype in water deficit conditions because of the impairment of stomatal closure and ROS production (Mustilli et al., 2002; Yoshida et al., 2006; Figure ).

Another crucial factor for stomatal closure is NO, which is generated in response to ABA (Neill et al., 2002, 2008). Exogenously applied NO donors triggered stomatal closure, whereas the application of an NO scavenger inhibited ABA-induced stomatal closure (Neill et al., 2002; Figure ).

There is some evidence that both H ₂O ₂ and NO actions in the guard cells require calcium. In addition, H ₂O ₂ inhibits K⁺ channel activity, induces cytosolic alkalization in the guard cells and promotes NO signaling in response to ABA (Zhang et al., 2001; Kohler et al., 2003; Wang and Song, 2008). Conversely, NO neither stimulates H ₂O ₂ synthesis nor does it require H ₂O ₂ for its action (Bright et al., 2006).

The Second Violin in the Concert of Stomatal Closure - The Role of Jasmonates in the Regulation of Stomatal Movement

Jasmonates are lipid-derived phytohormones that are involved in the regulation of vegetative and reproductive growth and the defense response against abiotic stress (Katsir et al., 2008). JA biosynthesis is induced by stress conditions (Wasternack, 2007) and many genes related to JA signaling are regulated by drought stress (Huang et al., 2008). The positive role of JA in the regulation of stomatal closure was observed in many studies (Gehring et al., 1997; Suhita et al., 2003, 2004; Munemasa et al., 2007). Similar to the ABA signaling pathway, JA signaling has been under intense investigation, particularly in relation to stress response. With the progress in research, many new components and their roles in JA-mediated stress response will be identified. Although the interaction between ABA and JA signaling pathways in stomata function has been established, there is still a need for further investigation and identification of the nodes linking these two signaling pathways, such as CPK6, which is described below.

When JA or methyl JA (MeJA) are applied exogenously to plants, they are converted into a biologically active form (+)-7-iso-Jasmonoyl-l-isoleucine (JA-Ile). JA-Ile is then bound by the receptor SCFCOI complex that contains the coronatine insensitive1 (COI1) F-box protein (Fonseca et al., 2009; Sheard et al., 2010). This interaction leads to the degradation of the repressor protein, JAZ (Jasmonate ZIM-domain), by the 26S proteasome and as a result, to the activation of distinct JA response genes by MYC2 (MYC domain transcription factor 2) (Chini et al., 2007; Thines et al., 2007; Fernández-Calvo et al., 2011). In the absence of JA, JAZ inhibits MYC2, which is then unable to activate the transcription of JA-inducible genes (Figure ).

Munemasa et al. ( 2011) identified CPK6 (Ca ²⁺ dependent protein kinase 6), which had previously been studied by Mori et al. ( 2006) in regards to ABA signaling, as a positive regulator of MeJA signaling in the guard cells. CDPKs function as important cytosolic Ca ²⁺ sensors in various plant physiological processes. Four kinases are involved in ABA signaling in Arabidopsis guard cells: CPK3, CPK6, CPK4, and CPK11; however, only mutations in the CPK6 impaired MeJA-induced stomatal closure (Munemasa et al., 2011). Like ABA, MeJA activates S-type anion channels. In coi1 ( coronatine insensitive 1) and cpk6 mutants, the activation of S-type anion channels was disrupted (Munemasa et al., 2007, 2011). Geiger et al. ( 2010) showed a direct interaction between CPK6 and the SLAC1 - S-type anion channel. The activation of SLAC1 by CPK6 was inhibited by the PP2Cs, ABI1, and ABI2, since abi1 and abi2 mutants exhibited insensitivity of stomata to MeJA, which leads to the inability of stomatal closure in response to MeJA (Figure ).

The formation of ROS and NO in the guard cells is not only induced by ABA and ethylene but also by MeJA. It has been showed that both ROS and NO levels were decreased in MeJA-insensitive mutants (Munemasa et al., 2007). Suhita et al. ( 2004) showed that a disruption of both genes encoding NADPH oxidase, AtrbohD and AtrbohF, results in the impairment of MeJA-induced stomatal closure and ROS production. However, in the cpk6 Arabidopsis mutant, ABA- and MeJA-mediated the production of ROS and NO was not reduced. CPK6 acts downstream of NO and ROS signaling and therefore may be a target of the NO-stimulated influx of Ca ²⁺ into the cytoplasm. As a feedback loop, MeJA-induced influx of Ca ²⁺ into the cytoplasm activates CPK6, which in turn is able to activate the S-type anion channel - SLAC1 (Figure ). This property of CPK6 makes it a node between the NO, ROS, ethylene and JA signaling pathways in ABA-induced stomatal closure (Munemasa et al., 2011; Figure ).

Jasmonates interacts with the ABA pathway by increasing the influx of Ca ²⁺, which stimulates CDPK and the resulting cascade in order to close stomata. Munemasa et al. ( 2007) reported that ABA or MeJA treatment triggers a reduction in the stomatal aperture within 10 min. MeJA-induced Ca ²⁺ levels were significantly lowered and stomatal closure was impaired when ABA biosynthesis inhibitors were added or when ABA-deficient mutants were studied. This suggests that jasmonate-induced changes in stomatal movements require endogenous ABA. In order to clarify this hypothesis, Hossain et al. ( 2011) examined the effect of 0.1 μM of ABA on MeJA-induced stomatal closure in aba 2-2 ( ABA deficient 2-2) mutants related to ABA biosynthesis. In the wild-type, 0.1 μM of ABA did not significantly induce either stomatal closure or Ca ²⁺ oscillations. The authors did not observe stomatal closure in aba2-2 when MeJA was applied without ABA, while in the presence of 0.1 μM ABA, MeJA induced stomatal closure.

When ABA Meets Ethylene

Ethylene is a gaseous phytohormone that is involved in the regulation of numerous plant processes such as seed germination, root-hair growth, leaf and flower senescence and abscission, fruit ripening, nodulation, and plant responses to stresses (Bleecker and Kende, 2000). It has been observed that ethylene can influence stomatal response via crosstalk with ABA; however, reports on its effect have been contradictory. Ethylene has been linked to the promotion of both stomatal closure (Pallas and Kays, 1982) and stomatal opening (Madhavan et al., 1983; Levitt et al., 1987; Merritt et al., 2001; Figure ). These contradictory effects need to be verified. One possible reason could be related to the methods used for stomatal observation that use detached leaves. Experiments with detached leaves do not always reflect the real response to stress or other applied factors in plants.

Tanaka et al. ( 2005) showed that Arabidopsis plants exposed to gaseous ethylene first did not close their stomata after the application of ABA. This was clear evidence that ethylene repressed ABA action in stomatal closure. In a drought stressed eto1 ( ethylene overproducer 1) mutant, stomata closed more slowly and were less sensitive to ABA than in the drought-treated wild type (Tanaka et al., 2005). In order to elucidate the interaction between ethylene and ABA during stomatal response, epidermal peels from the wild-type and eto1 were treated with ABA, ethylene, and both phytohormones. When ethylene was applied independently of ABA, it induced H ₂O ₂ synthesis within 30 min of the treatment. When ethylene was applied to the ABA-pretreated wild-type epidermal peels, an inhibition of stomatal closure was observed (Tanaka et al., 2005). Desikan et al. ( 2006) proved that ethylene-mediated stomatal closure is dependent on the H ₂O ₂ that is generated by NADPH oxidase. As was discussed above, H ₂O ₂ is one of the major molecules in ABA-induced stomatal closure.

There have been some studies that revealed both increased and decreased ethylene production in response to drought stress. However, most of them described experiments with detached leaves, which may not reflect the response of intact plants under drought conditions (Morgan et al., 1990; Abeles et al., 1992). Generally, elevated ABA concentrations limit the production of ethylene; and therefore a dramatic increase of ABA concentration during water stress probably causes a reduction in the production of ethylene (Sharp, 2002). The physiological mechanism of ethylene inhibition of the ABA-mediated stomatal closure may be related to the function of ethylene as a factor that ensures a minimum carbon dioxide supply for photosynthesis by keeping stomata half-opened under the stress conditions (Leung and Giraudat, 1998; Tanaka et al., 2005).

The-State-of-Art and Weak Points in Our Understanding of Stomatal Movements

Stomata are epidermal pores on a plant's surface that are essential for the control of water balance in plants. Many factors that are responsible for the regulation of stomatal movements have been already identified, such as components of ABA and other phytohormone signaling pathways. The most important, and one that is supported by well-documented studies, is the interaction between ABA (when taking into account its biosynthesis, catabolism, de/conjugation, and core signalosome) and the pumps and ion channels in the guard cell PM, in the regulation of stomatal movements under the stress.

However, further analyses of the networks of protein interactions, the co-expression of genes, metabolic factors, etc. should provide new insights into the key regulators of drought response in relation to guard cell movements. Taking into account that phytohormone pathways are still under intensive investigations and there are still many gaps to be elucidated, many of the already established interactions may be changed as further progress in research is achieved.

There are ambiguous reports in regards to the role of some phytohormones, such as ethylene, auxins, or cytokinins, in the regulation of stomatal movement that need to be clarified. In addition, the interaction between the diurnal cycle and ABA pathway should be further investigated in order to achieve a full understanding of this process.

There are some points that should be highlighted as a possible cause of the ambiguous reports related to the action of the regulators of stomatal movements. The first of these is the technique that is used to observe the stomata. Most analyses of stomata under stress are based on stomatal aperture observations. Some studies rely on stomata replicas from plants treated with stress and control, and observed under the light microscopy. This method is simple and inexpensive but generates problems due to the type of material used for the replicas. The accuracy and precision in the determination of stomatal aperture width is limited by the resolution of the standard light microscope. In contrast, scanning microscopy (SEM) offers high resolution images of stomata but requires expensive equipment and is not suitable for collecting large numbers of probes (Lawson et al., 1998). Recently, a popular technique in stomatal observations is confocal microscopy (Cañamero et al., 2006). As long as a proper technique that is not controversial in regards to its influence on stomatal response is not applied, all aperture measurements will be under discussion.

Another crucial problem is that most reports describe experiments with detached leaves, which may not reflect the response of intact plants under drought conditions (Morgan et al., 1990; Abeles et al., 1992; Dodd, 2012). Franks and Farquhar ( 2007) addressed the problem of data integration in stomatal research. They pointed out the lack of the integration of mechanical and quantitative physical information about guard cells and adjacent cells in model of stomatal function. Such integration of data should allow gas-exchange regulation to be better described and predicted. As long as guard cells are considered as a model without their surroundings, the results obtained may not be relevant. Another problem noted by Franks and Farquhar ( 2007) is that research on the impact of various environmental factors on the stomatal regulation and stomatal density should be performed on and compared among several species, not only one. This would allow a full picture of a broad morphological and evolutionary spectrum of possibilities of stomata development, density, and movement regulation in response to stresses to be obtained.

Summarizing, there are still many questions about the techniques used for evaluating the stomatal response to stress. Further development of proper methods will bring us closer to a fuller and more relevant understanding of stomatal action. The great progress in molecular biology studies enable insights into the signaling pathways, identification of new components, and interactions between them to be gained.

References

Abeles F. B., Morgan P. W., Saltveit M. E. (1992). Ethylene in Plant Biology. 2nd Edn. San Diego: Academic Press

Blatt M. R., Armstrong F. (1993). K⁺ channels of stomatal guard cells: abscisic-acid evoked control of the outward rectifier mediated by cytoplasmic pH. Planta 191, 330-34110.1007/BF00195690

Blatt M. R., Thiel G. (1994). K⁺ channels of stomatal guard cells: bimodal control of the K⁺ inward-rectifier evoked by auxin. Plant J. 5, 55-6810.1046/j.1365-313X.1994.5010055.x

Bright J., Desikan R., Hancock J. T., Weir I. S., Neill S. J. (2006). ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H₂O₂ synthesis. Plant J. 45, 113-12210.1111/j.1365-313X.2005.02615.x

Finkelstein R. R., Rock C. D. (2002). "Abscisic acid biosynthesis and response," in The Arabidopsis Book, eds Somerville C. R., Meyerowitz E. M., editors. (Rockville, MD: American Society of Plant Biologists; ), 1-52

Hosy E., Vavasseur A., Mouline K., Dreyer I., Gaymard F., Poree F., et al. (2003). The [Arabidopsis outward K⁺ channel GORK is involved in regulation of stomatal movements and plant transpiration. Proc. Natl. Acad. Sci. U.S.A. 100, 5549-555410.1073/pnas.0733970100PMC free article]

Hu H., Boisson-Dernier A., Israelsson-Nordström M., Böhmer M., Xue S., Ries A., et al. (2010). Carbonic anhydrases are upstream regulators in the guard cells of CO[₂-controlled stomatal movements. Nat. Cell Biol. 12, 87-9310.1038/ncb2009PMC free article]

Kagale S., Divi U. K., Krochko J. E., Keller W. A., Krishna P. (2007). Brassinosteroid confers tolerance in Arabidopsis thaliana and Brassica napus to a range of abiotic stresses. Planta 225, 353-36410.1007/s00425-006-0361-6

Kim T.-H., Bohmer M., Hu H., Nishimura N., Schroeder J. I. (2010). Guard cell signal transduction network: advances in understanding abscisic acid, CO[₂, and Ca²⁺ signaling. Annu. Rev. Plant Biol. 61, 561-59110.1146/annurev-arplant-042809-112226PMC free article]

Madhavan S., Chrmoinski A., Smith B. N. (1983). Effect of ethylene on stomatal opening in tomato and carnation leaves. Plant Cell Physiol. 24, 569-572

Marin E., Nussaume L., Quesada A., Gonneau M., Sotta B., Hugueney P., et al. (1996). Molecular identification of zeaxanthin epoxidase of [Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana. EMBO J. 15, 2331-2342PMC free article]

Melhorn V., Matsumi K., Koiwai H., Ikegami K., Okamoto M., Nambara E., et al. (2008). Transient expression of AtNCED3 and AAO3 genes in the guard cells causes stomatal closure in Vicia faba. J. Plant Res. 121, 125-13110.1007/s10265-007-0127-7

Pei Z. M., Kuchitsu K., Ward J. M., Schwarz M., Schroeder J. I. (1997). Differential abscisic acid regulation of guard cell slow anion channels in [Arabidopsis wild-type and abi1 and abi2 mutants. Plant Cell 9, 409-42310.2307/3870491PMC free article]

Raschke K., Shabahang M., Wolf R. (2003). The slow and the quick anion conductance in whole guard cells: their voltage-dependent alternation, and the modulation of their activities by abscisic acid and CO₂. Planta 217, 639-65010.1007/s00425-003-1034-3

Ueno K., Kinoshita T., Inoue S., Emi T., Shimazaki K. (2005). Biochemical characterization of plasma membrane H⁺-ATPase activation in guard cell protoplasts of Arabidopsis thaliana in response to blue light. Plant Cell Physiol. 46, 955-96310.1093/pcp/pci104

Willmer C., Fricker M. (1996). Stomata, 2nd Edn. London: Chapman & Hall.10.1007/978-94-011-0579-8

Xue S., Hu H., Ries A., Merilo E., Kollist H., Schroeder J. I. (2011). Central functions of bicarbonate in S-type anion channel activation and OST1 protein kinase in CO[₂ signal transduction in guard cell. EMBO J. 30, 1645-165810.1038/emboj.2011.68PMC free article]

Ye N., Zhu G., Liu Y., Li Y., Zhang J. (2011). ABA controls H₂O₂ accumulation through the induction of OsCATB in rice leaves under water stress. Plant Cell Physiol. 52, 689-69810.1093/pcp/pcr028

Zou J. J., Wei F.-J., Wang C., Wu J. J., Ratnasekera D., Li W.-X., et al. (2010). [Arabidopsis calcium-dependent protein kinase CPK10 functions in abscisic acid- and Ca²⁺-mediated stomatal regulation in response to drought. Plant Physiol. 154, 1232-124310.1104/pp.110.157545PMC free article]

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Open or Close the Gate - Stomata Action Under the Control of Phytohormones in Drought Stress Conditions

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Two highly specialized cells, the guard cells that surround the stomatal pore, are able to integrate environmental and endogenous signals in order to control the stomatal aperture and thereby the gas exchange. The uptake of CO[2] is associated with a ...

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mr i explains: The cause of Leaf Abscission (leaf drop)

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In this video, I explain how plant hormones Cytokinin, Auxin and Ethene are involved in the process of plant abscission. The video describes the abscission zone as being part of the petiole, the small stem connecting the leaf to the main stem, and highlights how cellulase enzymes break this region down following the withdrawal of nutrients.

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In the process of leaf abscission, plants periodically shed their leaves. Leaf abscission involves a number of biochemical and physical changes that are largely controlled by plant hormones.

Plants are primarily categorized as annuals, biennials, or perennials, based on their growth patterns. Annuals are those plants that undergo a complete life cycle from seed to seed in one growing season.

Biennials require two growing seasons to complete a life cycle; during the first year, only vegetative growth takes place. The above ground portion dies through the winter, and in the next growing season the roots send up a reproductive shoot that produces the seeds.

Perennials have the capacity to live through many successive growing seasons. In this group, those plants referred to as deciduous species shed all their leaves at the same time. The evergreen species shed leaves throughout the year, yet never shed the entire complement at any one time.

Preparation and Precursors

Prior to any natural abscission that may take place, leaves (or any other plant organ subject to abscission, such as flowers or fruit) undergo senescence. Senescence can be defined as the deterioration that occurs in conjunction with aging, and it results in the death of an organ or organism.

Senescence can occur throughout the entire plant, as it does in annuals, or in only the above ground portion, as it does in perennial herbs. In woody perennials, however, only the leaves senesce, while the bulk of the stem and roots remain alive.

From the time that leaves begin to grow, biochemical activities such as photosynthesis increase. This increase will continue until the leaves expand to maximum size. Soon after they reach maximum size, senescence begins, and photosynthetic rates begin to decrease. As the photosynthetic ability of the leaves declines, there is an accompanying decrease in other metabolic activities.

Respiration rates begin to subside dramatically, and leaf protein levels drop sharply because of increased proteolytic activity (enzymatic breakdown of proteins). Protein synthesis diminishes, and there is an increase in the enzymatic degradation of ribonucleic acid (RNA). There is also an increase in the hydrolytic breakdown of carbohydrates.

Finally, destruction of the green pigment, chlorophyll, is accompanied by increased visibility of the yellow or orange pigments called carotenoids, which were previously masked by chlorophyll. Most of the protein, carbohydrates, RNA, and chlorophyll degradation products are rapidly transported out of the senescing leaf. The final result is the production of yellowish, dead leaves.

The senescence process is a natural progression of the normal plant life cycle; however, environmental conditions can influence the process. Lack of water will speed the senescence process in most species. Higher-than-normal temperatures also cause an increase in senescence-related reactions. Darkness dramatically hastens senescence: Most leaves will senesce two or three times faster in darkness than if growing under normal light conditions.

Numerous studies strongly suggest that senescence is under hormonal control. Both ethylene and abscisic acid enhance senescence, but ethylene is the more effective of the two.

The gibberellins, cytokinins, and auxins (other types of hormones) have all been shown to delay the process in various plant species. The exact role of each of these hormones in senescence has not yet been determined, but it is apparent that the process involves the interaction of several of these growth-regulating substances.

Onset of Abscission

Following senescence, abscission of the leaves inevitably takes place. This process usually involves the formation of an abscission layer at the base of the leaf petiole.

During the early life of a leaf, auxin is produced in relatively high concentrations and is steadily transported out of the leaf through the petiole. As long as the auxin level remains high in the leaf and a sufficient amount of the hormone is transported across the petiole, both senescence and abscission are delayed.

In addition, gibberellins and cytokinins are produced elsewhere in the plant and then sent to the leaves to help retard the destructive processes. As the leaf matures, however, the level of the senescence-retarding hormones, especially auxin, decreases.

With the decrease in auxin levels, the catabolic (breakdown) reactions begin to outnumber the anabolic (synthetic) processes. In conjunction with the increase in catabolic reactions, there is a rise in the levels of abscisic acid and, especially, ethylene. Ethylene is particularly important in producing the abscission layer.

In most species, the abscission layer is formed from one or perhaps several layers of cells across the base of the petiole. In the earlier stages of abscission, there is a noticeable rise in the respiration rates of the cells of the abscission layer closest to the stem (the proximal cells). As the respiration rates increase to supply additional energy, ethylene stimulates one or more of these cell layers nearest the stem to increase in size.

Along with the increase in size of those cells, the cells in the abscission layer farthest from the stem (the distal cells) increase the production of enzymes that break down polysaccharides in the cell walls. With the secretion of these enzymes into the cell walls, digestion of the cell-wall materials begins.

The pressure created by the expansion of the cells in the proximal region of the abscission layer (causing them to grow against the weakened senescing cells of the distal region) results in the two layers breaking apart. Thus, the leaf detaches and falls from the plant.

Abscission, like senescence, is a natural order of progression during the life cycle of most plants. Although the process is closely correlated with regular seasonal changes, variations in environmental conditions can enhance abscission.

Deficiencies in certain nutrients, such as nitrogen, or lack of water can stimulate abscission. These conditions also hasten senescence, and senescence always precedes abscission. Hence, adverse environmental conditions perhaps trigger only the onset of senescence, and abscission occurs as a secondary result of the aging process.

Function of Abscission

Throughout much of the world, plants are subjected to freezing temperatures. The leaves of most plants are unable to withstand the cold weather and face certain death during the winter. If the leaves did not prepare for the onset of cold weather by undergoing senescence, the first freeze would kill the leaves before materials within them could be salvaged.

Without the abscission process to remove the dead leaves, the senescent tissues would shade the new spring growth that appears the following growing season. Hence, senescence and abscission provide a means by which perennials can recycle a major portion of leaf materials as the plants prepare for both the cold weather and the following growing season.

Competition for nutrients from other parts of the plant may initiate the senescence process. The pull of nutrients to another part of the plant such as roots, flowers, or fruit would reduce the amount of these materials bound for the leaves.

The reduced supply of nutrients could very well decrease synthetic rates, and the overall result would be a decline in major leaf macromolecules such as proteins, chlorophyll, and nucleic acids.

Competition alone, however, cannot account for the senescence and abscission phenomena, because even in plants that do not produce fruit, the leaves experience aging and the loss of leaves. In addition, numerous studies have shown that leaf senescence will still occur when flowers are removed from the plant soon after being formed.

Although competition for nutrients may not be the sole cause of the phenomenon, the mobilization of substances such as amino acids and carbohydrates from the leaves to other metabolic sinks, such as the fruit, is definitely linked to the initiation of senescence.

Several of the plant hormones or other factors that stimulate mobilization also hasten senescence. Hence, it is possible that the competition for nutrients triggers the production of some unknown senescence hormone by the fruit or some other competing plant part.

This theoretical substance would be transported to the leaves, where it would initiate mobilization of leaf contents. This mobilization might enhance senescence, which, in turn, might trigger the metabolic reactions that lead to abscission of the leaves.

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In the process of leaf abscission, plants periodically shed their leaves. Leaf abscission involves a number of biochemical and physical changes that are largely controlled by plant hormones. Plants are primarily categorized as annuals, biennials, or perennials, based on their growth patterns.

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In botany in reference to leaves, the term deciduous refers to a tree, shrub, or other plant that completely loses its foliage for part of the year.