Accumulation In Guard Cells Research Articles

Efficient gene editing of a model fern species through gametophyte-based transformation.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) system allows precise and easy editing of genes in many plant species. However, this system has not yet been applied to any fern species through gametophytes due to the complex characteristics of fern genomes, genetics, and physiology. Here, we established a protocol for gametophyte-based screening of single-guide RNAs (sgRNAs) with high efficiency for CRISPR/Cas9-mediated gene knockout in a model fern species, Ceratopteris richardii. We utilized the C. richardii ACTIN promoter to drive sgRNA expression and the enhanced CaMV 35S promoter to drive the expression of Streptococcus pyogenes Cas9 in this CRISPR-mediated editing system, which was employed to successfully edit a few genes, such as Nucleotidase/phosphatase 1 (CrSAL1) and Phytoene Desaturase (CrPDS), which resulted in an albino phenotype in C. richardii. Knockout of CrSAL1 resulted in significantly (P<0.05) reduced stomatal conductance (gs), leaf transpiration rate (E), guard cell length, and abscisic acid (ABA)-induced reactive oxygen species (ROS) accumulation in guard cells. Moreover, CrSAL1 overexpressing plants showed significantly increased net photosynthetic rate (A), gs, and E as well as most of the stomatal traits and ABA-induced ROS production in guard cells compared to the wild-type (WT) plants. Taken together, our optimized CRISPR/Cas9 system provides a useful tool for functional genomics in a model fern species, allowing the exploration of fern gene functions for evolutionary biology, herbal medicine discovery, and agricultural applications.

Arabidopsis Sucrose Synthase 3 (SUS3) regulates starch accumulation in guard cells at the end of day

ABSTRACT Starch in the stomatal guard cells is largely synthesized using carbon precursors originating from sugars imported from the leaf mesophyll. Such heterotrophic nature of guard cell starch synthesis prompted us to investigate the role of cytosolic sucrose synthases (SUS) in this pathway. Out of the six members of the Arabidopsis SUS gene family, SUS3 was the most highly expressed isoform in guard cells. The Arabidopsis sus3 mutant displayed changes in guard cell starch contents comparable to the Wild Type (WT) up until 6 h into the day. After this time point, sus3 guard cells surprisingly started to accumulate starch at very high rates, reaching the end of the day with significantly more starch than WT. Based on the phenotype of the sus3 mutant, we suggest that in guard cells, SUS3 is involved in the regulation of carbon fluxes towards starch synthesis during the second half of the day. SUS3 may be part of a previously predicted guard cell futile cycle of metabolic reactions, in which sucrose is re-synthesized from UDP-glucose to avoid excessive starch synthesis toward the end of the day. This is in contrast to typical storage organs, in which cytosolic SUS is required to produce ADP-glucose for starch synthesis.

The C2 H2 -type zinc finger transcription factor OSIC1 positively regulates stomatal closure under osmotic stress in poplar.

Salt and drought impair plant osmotic homeostasis and greatly limit plant growth and development. Plants decrease stomatal aperture to reduce water loss and maintain osmotic homeostasis, leading to improved stress tolerance. Herein, we identified the C2 H2 transcription factor gene OSMOTIC STRESS INDUCED C2 H2 1 (OSIC1) from Populus alba var. pyramidalis to be induced by salt, drought, polyethylene glycol 6000 (PEG6000) and abscisic acid (ABA). Overexpression of OSIC1 conferred transgenic poplar more tolerance to high salinity, drought and PEG6000 treatment by reducing stomatal aperture, while its mutant generated by the CRISPR/Cas9 system showed the opposite phenotype. Furthermore, OSIC1 directly up-regulates PalCuAOζ invitro and invivo, encoding a copper-containing polyamine oxidase, to enhance H2 O2 accumulation in guard cells and thus modulates stomatal closure when stresses occur. Additionally, ABA-, drought- and salt-induced PalMPK3 phosphorylates OSIC1 to increase its transcriptional activity to PalCuAOζ. This regulation of OSIC1 at the transcriptional and protein levels guarantees rapid stomatal closure when poplar responds to osmotic stress. Our results revealed a novel transcriptional regulatory mechanism of H2 O2 production in guard cells mediated by the OSIC1-PalCuAOζ module. These findings deepen our understanding of how perennial woody plants, like poplar, respond to osmotic stress caused by salt and drought and provide potential targets for breeding.

COP1 Mediates Dark-Induced Stomatal Closure by Suppressing FT, TSF and SOC1 Expression to Promote NO Accumulation in Arabidopsis Guard Cells

RING-finger-type ubiquitin E3 ligase Constitutively Photomorphogenic 1 (COP1) and floral integrators such as FLOWERING LOCUS T (FT), TWIN SISTER OF FT (TSF) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) have been identified as regulators of stomatal movement. However, little is known about their roles and relationship in dark-induced stomatal closure. Here, we demonstrated that COP1 is required for dark-induced stomatal closure using cop1 mutant. The cop1 mutant closed stomata in response to exogenous nitric oxide (NO) but not hydrogen peroxide (H2O2), and H2O2 but not NO accumulated in cop1 in darkness, further indicating that COP1 acts downstream of H2O2 and upstream of NO in dark-induced stomatal closure. Expression of FT, TSF and SOC1 in wild-type (WT) plants decreased significantly with dark duration time, but this process was blocked in cop1. Furthermore, ft, tsf, and soc1 mutants accumulated NO and closed stomata faster than WT plants in response to darkness. Altogether, our results indicate that COP1 transduces H2O2 signaling, promotes NO accumulation in guard cells by suppressing FT, TSF and SOC1 expression, and consequently leads to stomatal closure in darkness. These findings add new insights into the mechanisms of dark-induced stomatal closure.

Flavonoid-attracted Aeromonas sp. from the Arabidopsis root microbiome enhances plant dehydration resistance

Flavonoids are stress-inducible metabolites important for plant-microbe interactions. In contrast to their well-known function in initiating rhizobia nodulation in legumes, little is known about whether and how flavonoids may contribute to plant stress resistance through affecting non-nodulating bacteria. Here we show that flavonoids broadly contribute to the diversity of the Arabidopsis root microbiome and preferentially attract Aeromonadaceae, which included a cultivable Aeromonas sp. H1 that displayed flavonoid-induced chemotaxis with transcriptional enhancement of flagellum biogenesis and suppression of fumarate reduction for smooth swims. Strain H1 showed multiple plant-beneficial traits and enhanced plant dehydration resistance, which required flavonoids but not through a sudden “cry-for-help” upon stress. Strain H1 boosted dehydration-induced H2O2 accumulation in guard cells and stomatal closure, concomitant with synergistic induction of jasmonic acid-related regulators of plant dehydration resistance. These findings revealed a key role of flavonoids, and the underlying mechanism, in mediating plant-microbiome interactions including the bacteria-enhanced plant dehydration resistance.

Evolutionarily conserved bacterial effectors hijack abscisic acid signaling to induce an aqueous environment in the apoplast

High atmospheric humidity levels profoundly impact host-pathogen interactions in plants by enabling the establishment of an aqueous living space that benefits pathogens. The effectors HopM1 and AvrE1 of the bacterial pathogen Pseudomonas syringae have been shown to induce an aqueous apoplast under such conditions. However, the mechanisms by which this happens remain unknown. Here, we show that HopM1 and AvrE1 work redundantly to establish an aqueous living space by inducing a major reprogramming of the Arabidopsis thaliana transcriptome landscape. These effectors induce a strong abscisic acid (ABA) signature, which promotes stomatal closure, resulting in reduced leaf transpiration and water-soaking lesions. Furthermore, these effectors preferentially increase ABA accumulation in guard cells, which control stomatal aperture. Notably, a guard-cell-specific ABA transporter, ABCG40, is necessary for HopM1 induction of water-soaking lesions. This study provides molecular insights into a chain of events of stomatal manipulation that create an ideal microenvironment to facilitate infection.

Atmospheric pressure plasma treatment induces abscisic acid production, reduces stomatal aperture and improves seedling growth in Arabidopsis thaliana.

Cold atmospheric pressure plasmas (CAPPs) have been widely used for pre-sowing treatment in agriculture to accelerate seed germination; however, information on their application to pre-transplant seedlings is scarce. The roles of the phytohormone abscisic acid (ABA) on guard cell aperture that control air exchange with the environment were investigated after CAPPs treatment. In this study, Arabidopsis thaliana seedling growth was evaluated under CAPPs treatment at different doses. Besides, the optimal growth stimulation dose was selected to further evaluate changes in ABA, ROS, Ca2+ and stomatal aperture during growth .The expression of most ABA signalling genes were aslo examined to investigate the mechanism. CAPPs treatment for 1 min significantly promoted Arabidopsis seedling growth; the ABA concentration in seedlings increased and peaked 48 h after treatment but was lower than in the control after 96 h. Transcript levels of most ABA signalling genes were markedly enhanced at 48 h, although their transcripts were significantly downregulated after 96 h. CAPPs treatment also reduced stomatal aperture after 24 h and accelerated ROS accumulation in guard cells. The Ca2+ concentration in the treatment group was markedly higher than in the control at 24 and 96 h. The results suggest that CAPPs treatment accelerates ABA accumulation in Arabidopsis at early growth stages and ABA regulates ROS and Ca2+ concentrations to affect stomatal aperture, and both ABA and stoma size are affected in CAPPs stimulation of Arabidopsis seedling growth.

Ethylene and Sulfur Coordinately Modulate the Antioxidant System and ABA Accumulation in Mustard Plants under Salt Stress.

This study explored the interactive effect of ethephon (2-chloroethyl phosphonic acid; an ethylene source) and sulfur (S) in regulating the antioxidant system and ABA content and in maintaining stomatal responses, chloroplast structure, and photosynthetic performance of mustard plants (Brassica juncea L. Czern.) grown under 100 mM NaCl stress. The treatment of ethephon (200 µL L−1) and S (200 mg S kg−1 soil) together markedly improved the activity of enzymatic and non-enzymatic components of the ascorbate-glutathione (AsA-GSH) cycle, resulting in declined oxidative stress through lesser content of sodium (Na+) ion and hydrogen peroxide (H2O2) in salt-stressed plants. These changes promoted the development of chloroplast thylakoids and photosynthetic performance under salt stress. Ethephon + S also reduced abscisic acid (ABA) accumulation in guard cell, leading to maximal stomatal conductance under salt stress. The inhibition of ethylene action by norbornadiene (NBD) in salt- plus non-stressed treated plants increased ABA and H2O2 contents, and reduced stomatal opening, suggesting the involvement of ethephon and S in regulating stomatal conductance. These findings suggest that ethephon and S modulate antioxidant system and ABA accumulation in guard cells, controlling stomatal conductance, and the structure and efficiency of the photosynthetic apparatus in plants under salt stress.

PLDα1 and GPA1 are involved in the stomatal closure induced by Oridonin in Arabidopsis thaliana.

Oridonin is an important diterpenoid, which plays an important role in plant growth and development. PLDα1 and GPA1 are involved in many biotic or abiotic stresses. In this study, using the seedlings of Arabidopsis thaliana L. wild type (WT), PLDα1 defective mutant (pldα1), GPA1 defective mutant (gpa1) and pldα1/gpa1 double mutant as materials, the effect of stomatal apertures responding to Oridonin and the functions of PLDα1 and GPA1 in this response were investigated. The results showed that 60 μmol·L-1 of Oridonin induced stomatal closure and significantly increased the relative expression levels of GPA1 and PLDα1. Oridonin increased H2O2 accumulation in guard cells by inhibiting the antioxidant enzymes. The increase of H2O2 caused the expression of OST1, which is a positive regulatory gene for stomatal closure. Both PLDα1 and GPA1 were involved in Oridonin-induced stomatal closure and PLDα1 acted downstream of GPA1. The results suggested that Oridonin caused stomatal closure by affecting GPA1 and promoting PLDα1 to produce PA, and further accumulating H2O2 to upregulate gene OST1.

Two Chloroplast Proteins Negatively Regulate Plant Drought Resistance Through Separate Pathways.

Drought is one of the most deleterious environmental conditions affecting crop growth and productivity. Here we report the important roles of a nuclear-encoded chloroplast protein, PsbP Domain Protein 5 (PPD5), in drought resistance in Arabidopsis (Arabidopsis thaliana). From a forward genetic screen, a drought-resistant mutant named ppd5-2 was identified, which has a knockout mutation in PPD5 The ppd5 mutants showed increased H2O2 accumulation in guard cells and enhanced stomatal closure in response to drought stress. Further analysis revealed that the chloroplast-localized PPD5 protein interacts with and is phosphorylated by OST1, and phosphorylation of PPD5 increases its protein stability. Double mutant ppd5-2ost1-3 exhibited phenotypes resembling the ost1-3 single mutant with decreased stomatal closure, increased water loss, reduced H2O2 accumulation in guard cells, and hypersensitivity to drought stress. These results indicate that the chloroplast protein PPD5 negatively regulates drought resistance by modulating guard cell H2O2 accumulation via an OST1-dependent pathway. Interestingly, the thf1-1 mutant defective in the chloroplast protein THF1 displayed drought-resistance and H2O2 accumulation similar to the ppd5 mutants, but the thf1-1ost1-3 double mutant resembled the phenotypes of the thf1-1 single mutant. These results indicate that both OST1-dependent and OST1-independent pathways exist in the regulation of H2O2 production in chloroplasts of guard cells under drought stress conditions. Additionally, our findings suggest a strategy to improve plant drought resistance through manipulation of chloroplast proteins.

The Copper Amine Oxidase AtCuAOδ Participates in Abscisic Acid-Induced Stomatal Closure in Arabidopsis.

Plant copper amine oxidases (CuAOs) are involved in wound healing, defense against pathogens, methyl-jasmonate-induced protoxylem differentiation, and abscisic acid (ABA)-induced stomatal closure. In the present study, we investigated the role of the Arabidopsis thaliana CuAOδ (AtCuAOδ; At4g12290) in the ABA-mediated stomatal closure by genetic and pharmacological approaches. Obtained data show that AtCuAOδ is up-regulated by ABA and that two Atcuaoδ T-DNA insertional mutants are less responsive to this hormone, showing reduced ABA-mediated stomatal closure and H2O2 accumulation in guard cells as compared to the wild-type (WT) plants. Furthermore, CuAO inhibitors, as well as the hydrogen peroxide (H2O2) scavenger N,N1-dimethylthiourea, reversed most of the ABA-induced stomatal closure in WT plants. Consistently, AtCuAOδ over-expressing transgenic plants display a constitutively increased stomatal closure and increased H2O2 production compared to WT plants. Our data suggest that AtCuAOδ is involved in the H2O2 production related to ABA-induced stomatal closure.

Overexpression of TaLEA3 induces rapid stomatal closure under drought stress in Phellodendron amurense Rupr

Late embryogenesis abundant (LEA) proteins participate in drought stress responses in plants. In the present study, the gene TaLEA3 from the drought-resistant plant Tamarix androssowii was transformed into Amur cork tree (Phellodendron amurense) via Agrobacterium tumefaciens to investigate the mechanism of stomatal closure in response to osmotic stress. Our results showed that P. amurense overexpressing TaLEA3 were resistant to drought stress by rapid stomatal closure. To study the stomatal movement regulated at the molecular level, a model system for stoma closure was established in in vitro P. amurense. In this work, we found that the increased Ca2+ accumulation in guard cells of transgenic plants caused stomatal closure and activated K+ efflux under polyethylene glycol (PEG) stress. Moreover, H+ changes might provide a needed pH condition for stomatal closure. Further, nitric oxide (NO) fluorescence was measured using an NO-specific fluorescent probe, diaminofluorescein-FM diacetate, which showed that guard cell NO fluorescence was stronger in transgenic plants compared with wild type plants. Additionally, five genes encoding nitrate reductase were up-regulated, indicating that TaLEA3 overexpression positively regulated NO biosynthesis and accumulation in the guard cells. This discovery will further our understanding of the LEA gene function and will help in engineering drought-resistant tree cultivars.

Hydrogen sulfide may function downstream of hydrogen peroxide in mediating darkness-induced stomatal closure in Vicia faba.

The relationship between hydrogen sulfide (H2S) and hydrogen peroxide (H2O2) during darkness-induced stomatal closure in Vicia faba L. was investigated by using pharmacological, spectrophotographic and lasers canning confocal microscopic approaches. Darkness-induced stomatal closure was inhibited by H2S scavenger hypotaurine (HT), H2S synthesis inhibitors aminooxy acetic acid (AOA) and hydroxylamine (NH2OH) and potassium pyruvate (N3H3KO3) and ammonia (NH3), which are the products of L-/D-cysteine desulfhydrase (L-/D-CDes). Moreover, darkness induced H2S generation and increased L-/D-CDes activity in leaves of V. faba. H2O2 scavenger and synthesis inhibitors suppressed darkness-induced increase of H2S levels and L-/D-CDes activity as well as stomatal closure in leaves of V. faba. However, H2S scavenger and synthesis inhibitors had no effect on darkness-induced H2O2 accumulation in guard cells of V. faba. From these data it can be deduced that H2S is involved in darkness-induced stomatal closure and acts downstream of H2O2 in V. faba.

AgNO3 prevents the occurrence of hyperhydricity in Dianthus chinensis L. by enhancing water loss and antioxidant capacity

Hyperhydricity symptoms are common and significant during the in vitro culture of Dianthus chinensis L. and greatly affect the micropropagation and regeneration of cultured plantlets. However, effective measures for preventing such abnormalities have not been developed for this species. Silver nitrate (AgNO3) has been shown to revert hyperhydric plantlets to a normal state. Nevertheless, the effect of AgNO3 on the prevention of hyperhydricity and the underlying mechanisms remain unclear. In the present study, 98.7% of the Dianthus chinensis L. plantlets cultured in a hyperhydricity induction medium (HIM) developed symptoms of hyperhydricity; however, hyperhydricity symptoms were inhibited to different degrees when D. chinensis L. plantlets were cultured in HIM supplemented with various concentrations of AgNO3. In particular, approximately 97% of the D. chinensis L. plantlets grew normally and did not show any symptoms of hyperhydricity when cultured in HIM supplemented with 30 μmol L−1 AgNO3. Compared with the plantlets cultured in HIM alone, the plantlets cultured in HIM containing AgNO3 displayed dramatic decreases in water content, ethylene content, and reactive oxygen species (ROS) production (particularly regarding H2O2 accumulation in guard cells) and showed increased antioxidant enzyme activity, stoma aperture, and water loss. These changes not only prevented excess water from accumulating in the tissues of plantlets but also improved the antioxidant capacity of plantlets, ultimately resulting in the prevention of hyperhydricity.

Abscisic Acid-Induced Reactive Oxygen Species Are Modulated by Flavonols to Control Stomata Aperture.

Abscisic acid (ABA) increases reactive oxygen species (ROS) in guard cells to close Arabidopsis (Arabidopsis thaliana) stomata. In tomato (Solanum lycopersicum), we find that ABA-increased ROS is followed by stomatal closure and that both responses are blocked by inhibitors of ROS-producing respiratory burst oxidase enzymes. ABA-induced ROS sensor fluorescence accumulates in the nucleus, chloroplasts, and endomembranes. The accumulation of flavonol antioxidants in guard cells, but not surrounding pavement cells, was visualized by confocal microscopy using a flavonol-specific fluorescent dye. Decreased flavonols in guard cells in the anthocyanin reduced (are) mutant and elevated levels in the anthocyanin without (aw) mutant were quantified by confocal microscopy and in leaf extracts by mass spectrometry. Consistent with flavonols acting as antioxidants, higher levels of ROS were detected in guard cells of the tomato are mutant and lower levels were detected in aw both at homeostasis and after treatment with ABA. These results demonstrate the inverse relationship between flavonols and ROS. Guard cells of are show greater ABA-induced closure than the wild type, reduced light-dependent guard cell opening, and reduced water loss, with aw having opposite responses. Ethylene treatment of wild-type tomato plants increased flavonol accumulation in guard cells; however, no flavonol increases were observed in Neverripe (Nr), an ethylene receptor mutant. Consistent with lower levels of ROS due to elevated flavonols, ethylene treatments decreased ABA-induced stomatal closure in the wild type, but not Nr, with ethylene responses attenuated in the are mutant. Together, these results are consistent with flavonols dampening the ABA-dependent ROS burst that drives stomatal closure and facilitating stomatal opening to modulate leaf gas exchange.

ALA-Induced Flavonols Accumulation in Guard Cells Is Involved in Scavenging H2O2 and Inhibiting Stomatal Closure in Arabidopsis Cotyledons.

5-aminolevulinic acid (ALA), a new plant growth regulator, can inhibit stomatal closure by reducing H2O2 accumulation in guard cells. Flavonols are a main kind of flavonoids and have been proposed as H2O2 scavengers in guard cells. 5-aminolevulinic acid can significantly improve flavonoids accumulation in plants. However, whether ALA increases flavonols content in guard cells and the role of flavonols in ALA-regulated stomatal movement remains unclear. In this study, we first demonstrated that ALA pretreatment inhibited ABA-induced stomatal closure by reducing H2O2 accumulation in guard cells of Arabidopsis seedlings. This result confirms the inhibitory effect of ALA on stomatal closure and the important role of decreased H2O2 accumulation in this process. We also found that ALA significantly improved flavonols accumulation in guard cells using a flavonol-specific dye. Furthermore, using exogenous quercetin and kaempferol, two major components of flavonols in Arabidopsis leaves, we showed that flavonols accumulation inhibited ABA-induced stomatal movement by suppressing H2O2 in guard cells. Finally, we showed that the inhibitory effect of ALA on ABA-induced stomatal closure was largely impaired in flavonoid-deficient transparent testa4 (tt4) mutant. In addition, exogenous flavonols recovered stomatal responses of tt4 to the wild-type levels. Taken together, we conclude that ALA-induced flavonol accumulation in guard cells is partially involved in the inhibitory effect of ALA on ABA-induced H2O2 accumulation and stomatal closure. Our data provide direct evidence that ALA can regulate stomatal movement by improving flavonols accumulation, revealing new insights into guard cell signaling.

Flavonols Induced by 5-Aminolevulinic Acid Are Involved in Regulation of Stomatal Opening in Apple Leaves

Flavonols, the main flavonoids in plant leaves, have newly been proposed as a H2O2 scavenger in guard cells. However, whether 5-aminolevulinic acid (5-ALA) is involved in regulating stomatal movement of apple (Malus × domestica Borkh. ‘Fuji’) leaves through flavonol accumulation remains unclear. In this study, using diphenylboric acid 2-amino ethyl ester (DPBA, a flavonol fluorescent dye) and a laser scanning confocal microscope, we observed that 5-ALA pretreatment significantly improved flavonol accumulation in guard cells of apple leaves, especially around the nucleus. Then we demonstrated that 5-ALA pretreatment inhibited ABA-induced stomatal closure via decreasing reaction oxygen species (ROS) accumulation in guard cells. Pretreatment with quercetin or kaempferol, two main kinds of flavonols in plants, also inhibited ABA-induced stomatal closure by decreasing ROS content in the guard cells. Furthermore, exogenous flavonols could suppress H2O2-induced stomatal closure in apple leaves. Taken together, we conclude that ALA-induced flavonol accumulation in guard cells is involved in the inhibitory effect of ALA on ABA-induced ROS accumulation and stomatal closure in apple leaves.

Two Chloroplast Proteins Suppress Drought Resistance by Affecting ROS Production in Guard Cells.

Chloroplast as the site for photosynthesis is an essential organelle in plants, but little is known about its role in stomatal regulation and drought resistance. In this study, we show that two chloroplastic proteins essential for thylakoid formation negatively regulate drought resistance in Arabidopsis (Arabidopsis thaliana). By screening a mutant pool with T-DNA insertions in nuclear genes encoding chloroplastic proteins, we identified an HCF106 knockdown mutant exhibiting increased resistance to drought stress. The hcf106 mutant displayed elevated levels of reactive oxygen species (ROS) in guard cells, improved stomatal closure, and reduced water loss under drought conditions. The HCF106 protein was found to physically interact with THF1, a previously identified chloroplastic protein crucial for thylakoid formation. The thf1 mutant phenotypically resembled the hcf106 mutant and displayed more ROS accumulation in guard cells, increased stomatal closure, reduced water loss, and drought resistant phenotypes compared to the wild type. The hcf106thf1 double mutant behaved similarly as the thf1 single mutant. These results suggest that HCF106 and THF1 form a complex to modulate chloroplast function and that the complex is important for ROS production in guard cells and stomatal control in response to environmental stresses. Our results also suggest that modulating chloroplastic proteins could be a way for improving drought resistance in crops.

An NADPH-Oxidase/Polyamine Oxidase Feedback Loop Controls Oxidative Burst Under Salinity.

The apoplastic polyamine oxidase (PAO) catalyzes the oxidation of the higher polyamines spermidine and spermine, contributing to hydrogen peroxide (H2O2) accumulation. However, it is yet unclear whether apoplastic PAO is part of a network that coordinates the accumulation of reactive oxygen species (ROS) under salinity or if it acts independently. Here, we unravel that NADPH oxidase and apoplastic PAO cooperate to control the accumulation of H2O2 and superoxides (O2·-) in tobacco (Nicotiana tabacum). To examine to what extent apoplastic PAO constitutes part of a ROS-generating network, we examined ROS accumulation in guard cells of plants overexpressing or down-regulating apoplastic PAO (lines S2.2 and A2, respectively) or down-regulating NADPH oxidase (line AS-NtRbohD/F). The H2O2-specific probe benzene sulfonyl-H2O2 showed that, under salinity, H2O2 increased in S2.2 and decreased in A2 compared with the wild type. Surprisingly, the O2·--specific probe benzene sulfonyl-So showed that O2·- levels correlated positively with that of apoplastic PAO (i.e. showed high and low levels in S2.2 and A2, respectively). By using AS-NtRbohD/F lines and a pharmacological approach, we could show that H2O2 and O2·- accumulation at the onset of salinity stress was dependent on NADPH oxidase, indicating that NADPH oxidase is upstream of apoplastic PAO. Our results suggest that NADPH oxidase and the apoplastic PAO form a feed-forward ROS amplification loop, which impinges on oxidative state and culminates in the execution of programmed cell death. We propose that the PAO/NADPH oxidase loop is a central hub in the plethora of responses controlling salt stress tolerance, with potential functions extending beyond stress tolerance.

Starch Biosynthesis in Guard Cells But Not in Mesophyll Cells Is Involved in CO2-Induced Stomatal Closing.

Starch metabolism is involved in stomatal movement regulation. However, it remains unknown whether starch-deficient mutants affect CO2-induced stomatal closing and whether starch biosynthesis in guard cells and/or mesophyll cells is rate limiting for high CO2-induced stomatal closing. Stomatal responses to [CO2] shifts and CO2 assimilation rates were compared in Arabidopsis (Arabidopsis thaliana) mutants that were either starch deficient in all plant tissues (ADP-Glc-pyrophosphorylase [ADGase]) or retain starch accumulation in guard cells but are starch deficient in mesophyll cells (plastidial phosphoglucose isomerase [pPGI]). ADGase mutants exhibited impaired CO2-induced stomatal closure, but pPGI mutants did not, showing that starch biosynthesis in guard cells but not mesophyll functions in CO2-induced stomatal closing. Nevertheless, starch-deficient ADGase mutant alleles exhibited partial CO2 responses, pointing toward a starch biosynthesis-independent component of the response that is likely mediated by anion channels. Furthermore, whole-leaf CO2 assimilation rates of both ADGase and pPGI mutants were lower upon shifts to high [CO2], but only ADGase mutants caused impairments in CO2-induced stomatal closing. These genetic analyses determine the roles of starch biosynthesis for high CO2-induced stomatal closing.