Stomatal movements in response to environmental stimuli critically control the plant water status. Although these movements are governed by osmotically driven changes in guard cell volume, the role ...of membrane water channels (aquaporins) has remained hypothetical. Assays in epidermal peels showed that knockout Arabidopsis thaliana plants lacking the Plasma membrane Intrinsic Protein 2;1 (PIP2;1) aquaporin have a defect in stomatal closure, specifically in response to abscisic acid (ABA). ABA induced a 2-fold increase in osmotic water permeability (P
f) of guard cell protoplasts and an accumulation of reactive oxygen species in guard cells, which were both abrogated in pip2;1 plants. Open stomata 1 (OST1)/Snf1-related protein kinase 2.6 (SnRK2.6), a protein kinase involved in guard cell ABA signaling, was able to phosphorylate a cytosolic PIP2;1 peptide at Ser-121. OST1 enhanced PIP2;1 water transport activity when coexpressed in Xenopus laevis oocytes. Upon expression in pip2;1 plants, a phosphomimetic form (Ser121Asp) but not a phosphodeficient form (Ser121Ala) of PIP2;1 constitutively enhanced the P
f of guard cell protoplasts while suppressing its ABA-dependent activation and was able to restore ABA-dependent stomatal closure in pip2;1. This work supports a model whereby ABA-triggered stomatal closure requires an increase in guard cell permeability to water and possibly hydrogen peroxide, through OST1-dependent phosphorylation of PIP2;1 at Ser-121.
Vermiculite and micaceous minerals are relevant Cs
+
sorbents in soils and sediments. To understand the bioavailability of Cs
+
in soils resulting from multi-cation exchanges, sorption of Cs
+
onto ...clay minerals was performed in batch experiments with solutions containing Ca
2+
, Mg
2+
, and K
+
. A sequence between a vermiculite and various micaceous structures has been carried out by conditioning a vermiculite at various amounts of K. Competing cation exchanges were investigated as function of Cs
+
concentration. The contribution of K
+
on trace Cs
+
desorption is probed by applying different concentrations of K
+
on Cs-doped vermiculite and micaceous structures. Cs sorption isotherms at chemical equilibrium were combined with elemental mass balances in solution and structural analyses. Cs
+
replaces easily Mg
2+
> Ca
2+
and competes scarcely with K
+
. Cs
+
is strongly adsorbed on the various matrix, and a K/Cs ratio of about a thousand is required to remobilize Cs
+
. Cs
+
is exchangeable as long as the clay interlayer space remains open to Ca
2+
. However, an excess of K
+
, as well as Cs
+
, in solution leads to the collapse of the interlayer spaces that locks the Cs into the structure. Once K
+
and/or Cs
+
collapse the interlayer space, the external sorption sites are then particularly involved in Cs sorption. Subsequently, Cs
+
preferentially exchanges with Ca
2+
rather than Mg
2+
. Mg
2+
is extruded from the interlayer space by Cs
+
and K
+
adsorption, excluded from short interlayer space and replaced by Ca
2+
as Cs
+
desorbs.
Stomatal pores form a crucial interface between the leaf mesophyll and the atmosphere, controlling water and carbon balance in plants 1. Major advances have been made in understanding the regulatory ...networks and ion fluxes in the guard cells surrounding the stomatal pore 2. However, our knowledge on the role of carbon metabolism in these cells is still fragmentary 3–5. In particular, the contribution of starch in stomatal opening remains elusive 6. Here, we used Arabidopsis thaliana as a model plant to provide the first quantitative analysis of starch turnover in guard cells of intact leaves during the diurnal cycle. Starch is present in guard cells at the end of night, unlike in the rest of the leaf, but is rapidly degraded within 30 min of light. This process is critical for the rapidity of stomatal opening and biomass production. We exploited Arabidopsis molecular genetics to define the mechanism and regulation of guard cell starch metabolism, showing it to be mediated by a previously uncharacterized pathway. This involves the synergistic action of β-amylase 1 (BAM1) and α-amylase 3 (AMY3)—enzymes that are normally not required for nighttime starch degradation in other leaf tissues. This pathway is under the control of the phototropin-dependent blue-light signaling cascade and correlated with the activity of the plasma membrane H+-ATPase. Our results show that guard cell starch degradation has an important role in plant growth by driving stomatal responses to light.
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•Starch in guard cells is degraded within 30 min of light to promote stomatal opening•A distinct set of hydrolytic enzymes mediates starch degradation in guard cells•Phototropin signaling and proton pumping control starch degradation in guard cells•Guard cell starch drives plant growth through the control of stomatal aperture
Horrer et al. report quantitative analysis of starch turnover in guard cells of intact Arabidopsis leaves and show it to be an ideal system to study guard cell carbohydrate metabolism. Molecular genetic analyses show that guard cells integrate blue-light-induced proton pumping with starch degradation to control stomatal opening and plant growth.
The Arabidopsis (Arabidopsis thaliana) Heavy Metal Associated3 (AtHMA3) protein belongs to the P₁B₋₂ subgroup of the P-type ATPase family, which is involved in heavy metal transport. In a previous ...study, we have shown, using heterologous expression in the yeast Saccharomyces cerevisiae, that in the presence of toxic metals, AtHMA3 was able to phenotypically complement the cadmium/lead (Cd/Pb)-hypersensitive strain ycf1 but not the zinc (Zn)-hypersensitive strain zrc1. In this study, we demonstrate that AtHMA3 in planta is located in the vacuolar membrane, with a high expression level in guard cells, hydathodes, vascular tissues, and the root apex. Confocal imaging in the presence of the Zn/Cd fluorescent probe BTC-5N revealed that AtHMA3 participates in the vacuolar storage of Cd. A T-DNA insertional mutant was found more sensitive to Zn and Cd. Conversely, ectopic overexpression of AtHMA3 improved plant tolerance to Cd, cobalt, Pb, and Zn; Cd accumulation increased by about 2- to 3-fold in plants overexpressing AtHMA3 compared with wild-type plants. Thus, AtHMA3 likely plays a role in the detoxification of biological (Zn) and nonbiological (Cd, cobalt, and Pb) heavy metals by participating in their vacuolar sequestration, an original function for a P₁B₋₂ ATPase in a multicellular eukaryote.
Plant stomata function in innate immunity against bacterial invasion and abscisic acid (ABA) has been suggested to regulate this process. Using genetic, biochemical, and pharmacological approaches, ...we demonstrate that (i) the Arabidopsis thaliana nine-specific-lipoxygenase encoding gene, LOX1, which is expressed in guard cells, is required to trigger stomatal closure in response to both bacteria and the pathogen-associated molecular pattern flagellin peptide flg22; (ii) LOX1 participates in stomatal defense; (iii) polyunsaturated fatty acids, the LOX substrates, trigger stomatal closure; (iv) the LOX products, fatty acid hydroperoxides, or reactive electrophile oxylipins induce stomatal closure; and (v) the flg22-mediated stomatal closure is conveyed by both LOX1 and the mitogen-activated protein kinases MPK3 and MPK6 and involves salicylic acid whereas the ABA-induced process depends on the protein kinases OST1, MPK9, or MPK12. Finally, we show that the oxylipin and the ABA pathways converge at the level of the anion channel SLAC1 to regulate stomatal closure. Collectively, our results demonstrate that early biotic signaling in guard cells is an ABA-independent process revealing a novel function of LOX1-dependent stomatal pathway in plant immunity.
Reactive oxygen species (ROS) mediate abscisic acid (ABA) signaling in guard cells. To dissect guard cell ABA-ROS signaling genetically, a cell type-specific functional genomics approach was used to ...identify 2 MAPK genes, MPK9 and MPK12, which are preferentially and highly expressed in guard cells. To provide genetic evidence for their function, Arabidopsis single and double TILLING mutants that carry deleterious point mutations in these genes were isolated. RNAi-based gene-silencing plant lines, in which both genes are silenced simultaneously, were generated also. Mutants carrying a mutation in only 1 of these genes did not show any altered phenotype, indicating functional redundancy in these genes. ABA-induced stomatal closure was strongly impaired in 2 independent RNAi lines in which both MPK9 and MPK12 transcripts were significantly silenced. Consistent with this result, mpk9-1/12-1 double mutants showed an enhanced transpirational water loss and ABA- and H₂O₂-insensitive stomatal response. Furthermore, ABA and calcium failed to activate anion channels in guard cells of mpk9-1/12-1, indicating that these 2 MPKs act upstream of anion channels in guard cell ABA signaling. An MPK12-YFP fusion construct rescued the ABA-insensitive stomatal response phenotype of mpk9-1/12-1, demonstrating that the phenotype was caused by the mutations. The MPK12 protein is localized in the cytosol and the nucleus, and ABA and H₂O₂ treatments enhance the protein kinase activity of MPK12. Together, these results provide genetic evidence that MPK9 and MPK12 function downstream of ROS to regulate guard cell ABA signaling positively.
Abscisic acid (ABA) is a major phytohormone involved in important stress‐related and developmental plant processes. Recent phosphoproteomic analyses revealed a large set of ABA‐triggered ...phosphoproteins as putative mitogen‐activated protein kinase (MAPK) targets, although the evidence for MAPKs involved in ABA signalling is still scarce. Here, we identified and reconstituted in vivo a complete ABA‐activated MAPK cascade, composed of the MAP3Ks MAP3K17/18, the MAP2K MKK3 and the four C group MAPKs MPK1/2/7/14. In planta, we show that ABA activation of MPK7 is blocked in mkk3‐1 and map3k17mapk3k18 plants. Coherently, both mutants exhibit hypersensitivity to ABA and altered expression of a set of ABA‐dependent genes. A genetic analysis further reveals that this MAPK cascade is activated by the PYR/PYL/RCAR‐SnRK2‐PP2C ABA core signalling module through protein synthesis of the MAP3Ks, unveiling an atypical mechanism for MAPK activation in eukaryotes. Our work provides evidence for a role of an ABA‐induced MAPK pathway in plant stress signalling.
Plant mitogen-activated protein kinases (MAPKs) are involved in important processes, including stress signaling and development. In a functional yeast screen, we identified mutations that render ...Arabidopsis thaliana MAPKs constitutively active (CA). Importantly, CA-MAPKs maintain their specificity toward known activators and substrates. As a proof-of-concept, Arabidopsis MAPK4 (MPK4) function in plant immunity was investigated. In agreement with the phenotype of mpk4 mutants, CA-MPK4 plants were compromised in pathogen-induced salicylic acid accumulation and disease resistance. MPK4 activity was found to negatively regulate pathogen-associated molecular pattern-induced reactive oxygen species production but had no impact on callose deposition, indicating that CA-MPK4 allows discriminating between processes regulated by MPK4 activity from processes indirectly affected by mpk4 mutation. Finally, MPK4 activity was also found to compromise effector-triggered immunity conditioned by the Toll Interleukin-1 Receptor—nucleotide binding (NB)—Leu-rich repeat (LRR) receptors RPS4 and RPP4 but not by the coiled coil—NB-LRR receptors RPM1 and RPS2. Overall, these data reveal important insights on how MPK4 regulates plant defenses and establishes that CA-MAPKs offer a powerful tool to analyze the function of plant MAPK pathways.
Summary
In most plants, NO3− constitutes the major source of nitrogen, and its assimilation into amino acids is mainly achieved in shoots. Furthermore, recent reports have revealed that reduction of ...NO3− translocation from roots to shoots is involved in plant acclimation to abiotic stress. NPF2.3, a member of the NAXT (nitrate excretion transporter) sub‐group of the NRT1/PTR family (NPF) from Arabidopsis, is expressed in root pericycle cells, where it is targeted to the plasma membrane. Transport assays using NPF2.3‐enriched Lactococcus lactis membranes showed that this protein is endowed with NO3− transport activity, displaying a strong selectivity for NO3− against Cl−. In response to salt stress, NO3− translocation to shoots is reduced, at least partly because expression of the root stele NO3− transporter gene NPF7.3 is decreased. In contrast, NPF2.3 expression was maintained under these conditions. A loss‐of‐function mutation in NPF2.3 resulted in decreased root‐to‐shoot NO3− translocation and reduced shoot NO3− content in plants grown under salt stress. Also, the mutant displayed impaired shoot biomass production when plants were grown under mild salt stress. These mutant phenotypes were dependent on the presence of Na+ in the external medium. Our data indicate that NPF2.3 is a constitutively expressed transporter whose contribution to NO3− translocation to the shoots is quantitatively and physiologically significant under salinity.
Significance Statement
The constitutively expressed root stele transporter NPF2.3 from Arabidopsis promotes nitrate loading into the xylem sap and its contribution to nitrate allocation to shoots becomes quantitatively significant upon salt stress, which was unexpected because salinity is known to depress nitrate translocation. The loss‐of‐function mutation npf2.3 resulted in reduced biomass production when plants were grown in soil supplemented with sodium concentrations compatible with life cycle completion, indicating that NPF2.3 contributes to plant acclimation to moderate salinity.
Hydathodes are water pores found on leaves of a wide range of vascular plants and are the sites of guttation. We report here on the detailed anatomy of cauliflower (Brassica oleracea) and Arabidopsis ...(Arabidopsis thaliana) hydathodes. Hydathode surface presents pores resembling stomata giving access to large cavities. Beneath, the epithem is composed of a lacunar and highly vascularized parenchyma offering a direct connection between leaf surface and xylem vessels. Arabidopsis hydathode pores were responsive to ABA and light similar to stomata. The flg22 flagellin peptide, a well-characterized elicitor of plant basal immunity, did not induce closure of hydathode pores in contrast to stomata. Because hydathodes are natural infection routes for several pathogens, we investigated hydathode infection by the adapted vascular phytopathogenic bacterium Xanthomonas campestris pv campestris (Xcc), the causal agent of black rot disease of Brassicaceae. Microscopic observations of hydathodes six days postinoculation indicated a digestion of the epithem cells and a high bacterial multiplication. Postinvasive immunity was shown to limit pathogen growth in the epithem and is actively suppressed by the type III secretion system and its effector proteins. Altogether, these results give a detailed anatomic description of Brassicaceae hydathodes and highlight the efficient use of this tissue as an initial niche for subsequent vascular systemic dissemination of Xcc in distant plant tissues.
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