BACKGROUND
three hundred million hectares are irrigated worldwide.
secondary salinization of soils has become a major undesirable consequence of this agronomic activity.
with a greater need to increase crop yield on less productive land, a better knowledge of the physiological basis of salt tolerance will facilitate the engineering of salt tolerant crops needed to meet the near future food demand  <cit> .
nacl is the most soluble, widespread, and abundant of the salts in soils.
as low as 40 mm nacl generates an osmotic pressure of  <dig>  mpa and this stress manifests invariably as shoot growth arrest and senescence in most glycophytes  <cit> .
tolerance to salinity is commonly reflected in plant growth, which varies as the response progresses, and each phase of the adaptation may involve different signaling pathways  <cit> .
it is generally accepted that this response is biphasic comprised of a growth attenuating osmotic phase followed by a toxic ionic phase  <cit> .
the increased concentration of cytoplasmic sodium disrupts k+ homeostasis, affects general trans-membrane transport, and competes with mg2+ at the active site of many different enzymes, thus impairing metabolism  <cit>  manifesting as reduced cell division/expansion and increased senescence.
despite progress in understanding the phenomenon of plant salt-tolerance, the molecular basis for sensing and responding to extracellular na+ remains controversial  <cit> .
it is generally accepted that na+ sensing occurs via the salt overly sensitive  pathway  <cit> .
signaling is initiated in the cytoplasm by the sos2/sos <dig> calcium-responsive protein kinase pathway and transduced to the plasma membrane to regulate the na+/h+ antiporter  and reinstate ionic homeostasis.
however, there is still a poor understanding about the role of the plasma membrane  in sensing and signaling in response to na+  <cit> .
arguably, the best understood plasma membrane signaling pathway is mediated by the heterotrimeric g protein complex  <cit> .
in arabidopsis, this complex is comprised minimally of a gα subunit, a gβγ dimer and a 7-transmembrane  regulator of g signaling  protein .
genetic evidence supports a role for this g protein complex in glucose-stimulated cell proliferation and plant growth  <cit> .
in animals, a g protein-coupled receptor  catalyzes nucleotide exchange  on the gα subunit, leading to g activation.
in plants, the gα subunit spontaneously exchanges gdp for gtp without the requirement of a gpcr  <cit> .
therefore, to regulate g protein activity, most plants use a cell surface, 7-tm-rgs protein, the prototype being atrgs <dig>  <cit> .
atrgs <dig> keeps the complex in the inactive state.
sustained activation of g signaling involves physically uncoupling atrgs <dig> from the g protein complex to allow spontaneous nucleotide exchange and release of the gβγ dimer.
the cell accomplishes this uncoupling by endocytosis of atrgs <dig> whereby atrgs <dig> cycles through the endosome while the gtp-bound atgpa <dig> remains on the plasma membrane  <cit> .
few g protein-interacting elements that lie downstream of the activation step are known  <cit>  and none of the classic g protein targets from animals are found in plants  <cit> .
toward obtaining these downstream elements in plants, a g protein interactome was generated to assemble the set of plant-specific effectors using yeast complementation assays  <cit> .
this ab initio assembled g-protein interactome contains  <dig> interactions between  <dig> proteins .
among the various biological functions assigned to the 434 g-protein interactors, the response to salt stress function is overrepresented.
heterotrimeric g-protein signaling is indirectly linked with the salt response in plants other than arabidopsis.
for example, over-expression of gα or gβ genes obtained from pisum sativum confers increased tolerance to salt in transgenic tobacco  <cit> .
in rice, the steady-state levels of transcripts encoding the gα and gγ subunits are dramatically elevated by nacl but not kcl  <cit> .
indirect links between salt response and g-signaling can also be deduced for arabidopsis.
ablation of atwnk <dig> kinase, a key regulator of g signaling  <cit> , strengthens tolerance to salt an osmotic stress  <cit> .
gpa <dig> and agb <dig> mediate aba modulation of stomata aperture during stress  <cit> .
attenuation of growth and increased cell death by drought or salt is attributed, in part, to er stress  <cit> .
in arabidopsis, abg <dig> regulates the upr through an unknown pathway  <cit> .
here, we show that in response to na+, atrgs internalizes which is a robust indicator that the g-protein is activated.
as predicted from our functional analysis of g-protein interactors, genetic disruption of the g-signaling system results in plants with altered adaptation to salt stress.
loss of the gβγ dimer  confers hypersensitivity while loss of atrgs <dig> confers hyposensitivity to na+.
we propose a mechanism to balance growth and senescence under salt stress.
RESULTS
the g protein interactome suggests a role for g proteins in saline stress
additional file 1: data set s <dig>  shows that the “response to abiotic stimulus”, “response to stress”, and “metabolic process” are the top  <dig> most enriched go terms for g protein interactors.
a quarter of the detected plant-g-protein interactors were annotated as abiotic stimulus responsive proteins, and half are annotated as metabolism.
additional file 1: data set s <dig> is rank-ordered by p-values, consequently the top terms represent broader general annotations with less information.
we used the information contained in the resulting directed acyclic graph   <cit>  to systematize the selection of the most informative terms that are significantly enriched among interactors .
table  <dig> shows the  <dig> terms found at the terminal branches of the dag, all of them were found enriched with a corrected p-value <  <dig> .
by focusing on the terminal nodes, the annotations provided a clearer picture of the potential biological functions governed by g-proteins and their interactors.
*bh: multiple testing correction for p values .
the table includes go terms found in the terminals nodes of each branch in the directed acyclic graph.
these nodes contain the most specific and informative annotations that resulted significantly enriched.
enrichment  =   /  where n is the total number of genes associated to any go term, b is the total number of genes associated with a specific go term, n is the number of genes in the analyzed set, and b is the number of genes in the analyzed set associated to a particular go term.
table  <dig> reveals a combination of biotic and abiotic stress responses with central metabolic processes.
this suggests that a network of g protein interactors integrates nutrient availability with stress sensing to modulate growth and survival.
proteins with roles in osmotic and salt stress responses occupied one tenth of the g-interactome, and were enriched with the highest statistical support .
na+ activates plant g signaling
our functional profile analysis for the g-protein interactome suggested that g proteins mediate nacl responses.
to test this hypothesis, we transplanted 5-d-old arabidopsis seedlings of the different genotypes from ¼ ms agar plates to ¼ ms agar plates supplemented with 200 mm nacl.
arabidopsis seedlings lacking the gβ subunit of the heterotrimeric g protein complex  rapidly senesced  compared to the wild-type.
agb1- <dig> seedlings became bleached of chlorophyll while col- <dig> seedlings displayed typical stress symptoms such as high levels of anthocyanin  but did not bleach.
this prompted the hypothesis that nacl itself directly or indirectly activates g signaling to promote stress survival.
to test activation, plants expressing atrgs1-yfp were treated with nacl or kcl and atrgs1-yfp internalization was quantitated.
atrgs <dig> internalization is a standard reporter for g protein activation  <cit> .
nacl, but not kcl, initiated g signaling indicating activation is caused by na+ not cl− .
proteins visualized in the endosome after nacl treatment had a plasma membrane origin since blocking new synthesis of protein had no effect on the subcellular location after treatment .
atrgs and agb <dig>  components of the same g protein complex, have antagonistic roles in the survival of arabidopsis to salt stress
plants lacking atrgs <dig>  or the gβ subunit , which is required for activation of g-signaling  <cit> , showed clear differences in shoot growth when germinated and grown on ¼ ms agar media supplemented with nacl .
attenuation of shoot growth and hastening of leaf senescence are well-characterized phenotypes displayed by plants grown under saline stress.
compared with col- <dig>  agb1- <dig> seedlings on nacl were small and chlorotic.
in contrast, rgs1- <dig> mutants were larger and less chlorotic than col- <dig>  the accelerated senescence observed in agb1- <dig> seedlings growing in nacl-supplemented agar plates was also observed when plants were grown on soil .
agb1- <dig> mutants showed clear chlorotic lesions in older leaves possibly due to higher accumulation of na+ in this tissue.
arabidopsis is a glycophyte.
at moderate salt concentration , growth is already noticeably affected and at 100 mm nacl growth is severely inhibited.
as clearly evident with the hypersensitive agb1- <dig> genotype, plant development is arrested and almost all the seedlings die at early stages .
the differential sensitivities between col <dig>  agb1- <dig> and rgs1- <dig> were tested under different concentrations of nacl .
the greatest difference among the genotypes was at 100 mm nacl.
the ability of 100 mm nacl to arrest development was used to develop a “greening assay” to quantitate the salt-induced phenotypes of g-protein mutants .
green seedlings that were not arrested were scored and the percent indicated for each treatment.
rgs1- <dig> mutants had fewer arrested seedlings compared to col- <dig>  in contrast to agb1- <dig> seedlings which were almost all arrested .
these effects were not observed with the same concentration of kcl indicating that the observed phenotype is na+ sodium specific .
an osmotic response is ruled out since an equal osmotic pressure applied with mannitol had no effect on shoots of the different genotypes tested .
seedlings lacking the gα subunit  behaved similarly to rgs <dig> null mutants  consistent with atrgs <dig> signaling operating through its cognate gα subunit .
it also suggests that the primary signaling element is the gβγ dimer since loss of either atrgs <dig> or atgpa <dig> increases the pool size of freed gβγ dimer at the plasma membrane.
as expected, when all three of the gγ subunits are genetically deleted thus removing agb <dig> from the plasma membrane, plants had the agb1- <dig> phenotype.
loss of either agg <dig> or agg <dig> had little or no effect suggesting functional redundancy or that agb <dig> dimers comprised with agg <dig> or agg <dig> are not involved in the na+ response.
the agb1- <dig> allele was epistatic to the rgs1- <dig> allele consistent with agb <dig> acting downstream of atrgs <dig> .
like rgs1- <dig> mutants, fewer gpa1- <dig> seedlings were arrested on 100 mm nacl compared to col- <dig>  however they were 30% smaller than rgs1- <dig> , thus the gpa <dig> “salt” phenotype is not exactly like the rgs <dig> phenotype.
endocytosis of atrgs <dig> causes sustained activation of the gα subunit and the gβγ dimer at the plasma membrane and this process requires phosphorylation by wnk <dig> kinase  <cit> .
interestingly, mammalian homologs of plant wnk <dig> regulate na+/k+ channel activity through a signaling phosphorylation pathway involving oxidative stress responsive kinases  <cit> .
zhang and coworkers reported that loss of wnk <dig> conferred salt tolerance  <cit> .
since wnk <dig> is required for atrgs <dig> endocytosis, we expected that combining loss-of-function mutations in both atrgs <dig> and wnk <dig> would be epistatic.
however, loss of both atrgs <dig> and wnk <dig>  conferred slightly more nacl tolerance than the rgs1- <dig> allele alone suggesting a small additive effect .
elevating active, plasma membrane atgpa <dig> subunit and agb1/agg dimer conferred salt tolerance
to test whether atrgs <dig> operates through the gα subunit to regulate the activity of the gβγ dimer, we used two point mutations that independently disrupt binding between atgpa <dig> and agb <dig> and consequently increase the pool of activated g proteins at the plasma membrane  <cit> .
disrupting heterotrimer formation is expected to increase the pool of active gα and gβγ subunits without disrupting atrgs <dig> function.
mutant agb <dig> proteins were expressed in the agb1- <dig> null background and at least two independent lines were characterized.
both w <dig> and s <dig> residues lie within the gα-gβ protein interface and mutation of these residues to alanine prevents gα binding to gβγ without disruption of the plasma membrane localization  <cit> .
reduced heterotrimer formation means an increase in activated gα subunit and gβγ dimer at the plasma membrane.
mutations in these residues confer nacl tolerance .
the positive control was a set of mutations on the surface located outside the gα-gβ surface of interaction .
this mutant agb <dig> rescued the agb1- <dig> null mutant to wild type levels of tolerance.
the negative control was a set of mutations in a surface patch  known to be involved in many cellular responses  <cit> .
this mutant agb <dig> did not rescue the agb1- <dig> salt-sensitive phenotype.
g signaling mediates glucose-induced tolerance to nacl
a regulatory signaling network integrating environmental cues with nutritional status may play a key role in shunting energy from developmental-linked biosynthetic metabolism into metabolic pathways aimed at boosting stress tolerance  <cit> .
since both atrgs <dig> and agb <dig> are part of a sugar-dependent signaling pathway and corresponding mutants have altered responsiveness to nacl, we tested if sugar sensing was a factor of the nacl response.
sucrose  and glucose , improved salt tolerance for col- <dig> seedlings.
glucose had no effect on salt tolerance for rgs1- <dig> or agb1- <dig> mutants  at the tested concentration.
discussion
geng et al.
<cit>  elegantly showed that the reaction of a root to salt is complex and has dynamics in both temporal and spatial dimensions.
upon an initial shock to applied salt, the root growth rate dramatically decreases within the first few hours  followed by a slow constant growth rate over the next few hours .
during the next ~10 hours, growth recovers  albeit not to the full rate of the control roots and then growth reaches a new steady-state rate .
this implies a complex and dynamic regulatory system.
indeed, geng, et al.
<cit>  showed that throughout this timeline different transcriptional programs begin and end in a tissue-specific context.
given the delay in activation , g proteins are most likely to be involved in the recovery phase but the mechanism is unclear and at this juncture, we can only speculate based on the observations that we and others report.
in many plants, the 7tm-rgs protein holds the gα subunit loaded with gdp in an inactive state, which favors the formation of the inactive heterotrimer .
because plant gα subunits spontaneously exchange gdp for gtp, in the absence of the 7tm-rgs, the gα subunit is gtp bound  and the gβγ dimer is freed.
sustained activation of g signaling involves physically uncoupling 7tm-rgs from the g protein complex to allow spontaneous nucleotide exchange and release of the gβγ dimer.
the cell accomplishes this physical uncoupling by endocytosis of the 7tm-rgs whereby it cycles through the endosome while the gtp-bound gα subunit remains on the plasma membrane  <cit> .
it is abundantly clear that endocytosis of atrgs <dig> increases the active g protein pool  <cit> .
upon activation, the gtp-bound gα subunit releases the gβγ dimer enabling both g protein components to interact with cellular targets.
the gα subunit is a positive modulator of cell proliferation in arabidopsis  <cit>  and the gβγ dimer, among other roles it plays, operates in the upr, which is important for cell survival during er stress  <cit> .
salt induces the upr in which case the upr alleviates nacl stress by increasing er-associated protein degradation  <cit> .
failure to do so triggers salt-associated-cell-death  <cit> .
agb <dig> plays a positive role in the upr response  <cit> , since three tested loss-of-function agb <dig> mutants showed hypersensitivity to tunicamycin   <cit> .
the genetic data here support this biological context.
in the rgs1- <dig> mutant, two important activities are increased at the plasma membrane: a proliferative factor  and an er-stress reliever factor .
consequently, mutations that promote active gα subunit and gβγ dimers confer sustained growth and tolerance to nacl compared to wt.
consistent with this idea, disruption of the heterotrimer in a way that promotes active atgpa <dig> and agb <dig> without the loss of atrgs <dig> also conferred tolerance to nacl compared to col- <dig> .
the gpa1- <dig> phenotype is also consistent with this conclusion; in the absence of atgpa <dig>  plants have more activated agb <dig> at the plasma membrane as for loss of atrgs <dig>  therefore these mutants are less sensitive to the stress.
however, gpa <dig> mutants lack the cell proliferation function, therefore they do not behave phenotypically exactly like rgs <dig> mutants .
in contrast, agb1- <dig> and agb1-2/ rgs1- <dig> double mutants lack gβ thus making these seedlings highly sensitive to the er-stress imposed by nacl.
while it is possible that atrgs <dig> senses na+, we do not favor this view since the nacl effect  is slow, thus activation may be indirect through an increase in glucose by regulation of glucose metabolism enzymes leading to increased sugar levels.
in fact, it was clearly demonstrated that nacl increases sugar levels in root  <cit>  and leaf cells  <cit> .
this also explains the ameliorative effect of applied glucose on nacl responsiveness .
CONCLUSIONS
plant tolerance to nacl, in particular the recovery phase, involves the plasma membrane g protein-mediated glucose-signaling pathway.
the mechanism for survival to salt stress requires g protein activation by releasing freed gα subunits and gβγ dimers.
the discovery here of sodium-induced activation of g signaling via atrgs <dig> endocytosis, whether or not direct or indirect through increased glucose levels, raises further complexity involving feedback loops that will need to be addressed.

 three hundred million hectares are irrigated worldwide. secondary salinization of soils has become a major undesirable consequence of this agronomic activity. with a greater need to increase crop yield on less productive land, a better knowledge of the physiological basis of salt tolerance will facilitate the engineering of salt tolerant crops needed to meet the near future food demand. nacl is the most soluble, widespread, and abundant of the salts in soils. as low as 40 mm nacl generates an osmotic pressure of  mpa and this stress manifests invariably as shoot growth arrest and senescence in most glycophytes. tolerance to salinity is commonly reflected in plant growth, which varies as the response progresses, and each phase of the adaptation may involve different signaling pathways. it is generally accepted that this response is biphasic comprised of a growth attenuating osmotic phase followed by a toxic ionic phase. the increased concentration of cytoplasmic sodium disrupts k+ homeostasis, affects general trans-membrane transport, and competes with mg2+ at the active site of many different enzymes, thus impairing metabolism manifesting as reduced cell division/expansion and increased senescence. despite progress in understanding the phenomenon of plant salt-tolerance, the molecular basis for sensing and responding to extracellular na+ remains controversial. it is generally accepted that na+ sensing occurs via the salt overly sensitive pathway. signaling is initiated in the cytoplasm by the sos2/sos calcium-responsive protein kinase pathway and transduced to the plasma membrane to regulate the na+/h+ antiporter and reinstate ionic homeostasis. however, there is still a poor understanding about the role of the plasma membrane in sensing and signaling in response to na+ . arguably, the best understood plasma membrane signaling pathway is mediated by the heterotrimeric g protein complex. in arabidopsis, this complex is comprised minimally of a gα subunit, a gβγ dimer and a 7-transmembrane regulator of g signaling protein. genetic evidence supports a role for this g protein complex in glucose-stimulated cell proliferation and plant growth. in animals, a g protein-coupled receptor catalyzes nucleotide exchange on the gα subunit, leading to g activation. in plants, the gα subunit spontaneously exchanges gdp for gtp without the requirement of a gpcr. therefore, to regulate g protein activity, most plants use a cell surface, 7-tm-rgs protein, the prototype being atrgs. atrgs keeps the complex in the inactive state. sustained activation of g signaling involves physically uncoupling atrgs from the g protein complex to allow spontaneous nucleotide exchange and release of the gβγ dimer. the cell accomplishes this uncoupling by endocytosis of atrgs whereby atrgs cycles through the endosome while the gtp-bound atgpa remains on the plasma membrane. few g protein-interacting elements that lie downstream of the activation step are known and none of the classic g protein targets from animals are found in plants. toward obtaining these downstream elements in plants, a g protein interactome was generated to assemble the set of plant-specific effectors using yeast complementation assays. this ab initio assembled g-protein interactome contains interactions between proteins. among the various biological functions assigned to the 434 g-protein interactors, the response to salt stress function is overrepresented. heterotrimeric g-protein signaling is indirectly linked with the salt response in plants other than arabidopsis. for example, over-expression of gα or gβ genes obtained from pisum sativum confers increased tolerance to salt in transgenic tobacco. in rice, the steady-state levels of transcripts encoding the gα and gγ subunits are dramatically elevated by nacl but not kcl. indirect links between salt response and g-signaling can also be deduced for arabidopsis. ablation of atwnk kinase, a key regulator of g signaling, strengthens tolerance to salt an osmotic stress. gpa and agb mediate aba modulation of stomata aperture during stress. attenuation of growth and increased cell death by drought or salt is attributed, in part, to er stress. in arabidopsis, abg regulates the upr through an unknown pathway. here, we show that in response to na+, atrgs internalizes which is a robust indicator that the g-protein is activated. as predicted from our functional analysis of g-protein interactors, genetic disruption of the g-signaling system results in plants with altered adaptation to salt stress. loss of the gβγ dimer confers hypersensitivity while loss of atrgs confers hyposensitivity to na+. we propose a mechanism to balance growth and senescence under salt stress.  the g protein interactome suggests a role for g proteins in saline stress additional file 1: data set s shows that the “response to abiotic stimulus”, “response to stress”, and “metabolic process” are the top most enriched go terms for g protein interactors. a quarter of the detected plant-g-protein interactors were annotated as abiotic stimulus responsive proteins, and half are annotated as metabolism. additional file 1: data set s is rank-ordered by p-values, consequently the top terms represent broader general annotations with less information. we used the information contained in the resulting directed acyclic graph to systematize the selection of the most informative terms that are significantly enriched among interactors. table  shows the terms found at the terminal branches of the dag, all of them were found enriched with a corrected p-value < . by focusing on the terminal nodes, the annotations provided a clearer picture of the potential biological functions governed by g-proteins and their interactors. *bh: multiple testing correction for p values. the table includes go terms found in the terminals nodes of each branch in the directed acyclic graph. these nodes contain the most specific and informative annotations that resulted significantly enriched. enrichment  =   /  where n is the total number of genes associated to any go term, b is the total number of genes associated with a specific go term, n is the number of genes in the analyzed set, and b is the number of genes in the analyzed set associated to a particular go term. table  reveals a combination of biotic and abiotic stress responses with central metabolic processes. this suggests that a network of g protein interactors integrates nutrient availability with stress sensing to modulate growth and survival. proteins with roles in osmotic and salt stress responses occupied one tenth of the g-interactome, and were enriched with the highest statistical support. na+ activates plant g signaling our functional profile analysis for the g-protein interactome suggested that g proteins mediate nacl responses. to test this hypothesis, we transplanted 5-d-old arabidopsis seedlings of the different genotypes from ¼ ms agar plates to ¼ ms agar plates supplemented with 200 mm nacl. arabidopsis seedlings lacking the gβ subunit of the heterotrimeric g protein complex rapidly senesced compared to the wild-type. agb1- seedlings became bleached of chlorophyll while col- seedlings displayed typical stress symptoms such as high levels of anthocyanin but did not bleach. this prompted the hypothesis that nacl itself directly or indirectly activates g signaling to promote stress survival. to test activation, plants expressing atrgs1-yfp were treated with nacl or kcl and atrgs1-yfp internalization was quantitated. atrgs internalization is a standard reporter for g protein activation. nacl, but not kcl, initiated g signaling indicating activation is caused by na+ not cl−. proteins visualized in the endosome after nacl treatment had a plasma membrane origin since blocking new synthesis of protein had no effect on the subcellular location after treatment. atrgs and agb components of the same g protein complex, have antagonistic roles in the survival of arabidopsis to salt stress plants lacking atrgs or the gβ subunit, which is required for activation of g-signaling, showed clear differences in shoot growth when germinated and grown on ¼ ms agar media supplemented with nacl. attenuation of shoot growth and hastening of leaf senescence are well-characterized phenotypes displayed by plants grown under saline stress. compared with col- agb1- seedlings on nacl were small and chlorotic. in contrast, rgs1- mutants were larger and less chlorotic than col- the accelerated senescence observed in agb1- seedlings growing in nacl-supplemented agar plates was also observed when plants were grown on soil. agb1- mutants showed clear chlorotic lesions in older leaves possibly due to higher accumulation of na+ in this tissue. arabidopsis is a glycophyte. at moderate salt concentration, growth is already noticeably affected and at 100 mm nacl growth is severely inhibited. as clearly evident with the hypersensitive agb1- genotype, plant development is arrested and almost all the seedlings die at early stages. the differential sensitivities between col agb1- and rgs1- were tested under different concentrations of nacl. the greatest difference among the genotypes was at 100 mm nacl. the ability of 100 mm nacl to arrest development was used to develop a “greening assay” to quantitate the salt-induced phenotypes of g-protein mutants. green seedlings that were not arrested were scored and the percent indicated for each treatment. rgs1- mutants had fewer arrested seedlings compared to col- in contrast to agb1- seedlings which were almost all arrested. these effects were not observed with the same concentration of kcl indicating that the observed phenotype is na+ sodium specific. an osmotic response is ruled out since an equal osmotic pressure applied with mannitol had no effect on shoots of the different genotypes tested. seedlings lacking the gα subunit behaved similarly to rgs null mutants consistent with atrgs signaling operating through its cognate gα subunit. it also suggests that the primary signaling element is the gβγ dimer since loss of either atrgs or atgpa increases the pool size of freed gβγ dimer at the plasma membrane. as expected, when all three of the gγ subunits are genetically deleted thus removing agb from the plasma membrane, plants had the agb1- phenotype. loss of either agg or agg had little or no effect suggesting functional redundancy or that agb dimers comprised with agg or agg are not involved in the na+ response. the agb1- allele was epistatic to the rgs1- allele consistent with agb acting downstream of atrgs. like rgs1- mutants, fewer gpa1- seedlings were arrested on 100 mm nacl compared to col- however they were 30% smaller than rgs1-, thus the gpa “salt” phenotype is not exactly like the rgs phenotype. endocytosis of atrgs causes sustained activation of the gα subunit and the gβγ dimer at the plasma membrane and this process requires phosphorylation by wnk kinase. interestingly, mammalian homologs of plant wnk regulate na+/k+ channel activity through a signaling phosphorylation pathway involving oxidative stress responsive kinases. zhang and coworkers reported that loss of wnk conferred salt tolerance. since wnk is required for atrgs endocytosis, we expected that combining loss-of-function mutations in both atrgs and wnk would be epistatic. however, loss of both atrgs and wnk conferred slightly more nacl tolerance than the rgs1- allele alone suggesting a small additive effect. elevating active, plasma membrane atgpa subunit and agb1/agg dimer conferred salt tolerance to test whether atrgs operates through the gα subunit to regulate the activity of the gβγ dimer, we used two point mutations that independently disrupt binding between atgpa and agb and consequently increase the pool of activated g proteins at the plasma membrane. disrupting heterotrimer formation is expected to increase the pool of active gα and gβγ subunits without disrupting atrgs function. mutant agb proteins were expressed in the agb1- null background and at least two independent lines were characterized. both w and s residues lie within the gα-gβ protein interface and mutation of these residues to alanine prevents gα binding to gβγ without disruption of the plasma membrane localization. reduced heterotrimer formation means an increase in activated gα subunit and gβγ dimer at the plasma membrane. mutations in these residues confer nacl tolerance. the positive control was a set of mutations on the surface located outside the gα-gβ surface of interaction. this mutant agb rescued the agb1- null mutant to wild type levels of tolerance. the negative control was a set of mutations in a surface patch known to be involved in many cellular responses. this mutant agb did not rescue the agb1- salt-sensitive phenotype. g signaling mediates glucose-induced tolerance to nacl a regulatory signaling network integrating environmental cues with nutritional status may play a key role in shunting energy from developmental-linked biosynthetic metabolism into metabolic pathways aimed at boosting stress tolerance. since both atrgs and agb are part of a sugar-dependent signaling pathway and corresponding mutants have altered responsiveness to nacl, we tested if sugar sensing was a factor of the nacl response. sucrose and glucose, improved salt tolerance for col- seedlings. glucose had no effect on salt tolerance for rgs1- or agb1- mutants at the tested concentration. discussion geng et al. elegantly showed that the reaction of a root to salt is complex and has dynamics in both temporal and spatial dimensions. upon an initial shock to applied salt, the root growth rate dramatically decreases within the first few hours followed by a slow constant growth rate over the next few hours. during the next ~10 hours, growth recovers albeit not to the full rate of the control roots and then growth reaches a new steady-state rate. this implies a complex and dynamic regulatory system. indeed, geng, et al. showed that throughout this timeline different transcriptional programs begin and end in a tissue-specific context. given the delay in activation, g proteins are most likely to be involved in the recovery phase but the mechanism is unclear and at this juncture, we can only speculate based on the observations that we and others report. in many plants, the 7tm-rgs protein holds the gα subunit loaded with gdp in an inactive state, which favors the formation of the inactive heterotrimer. because plant gα subunits spontaneously exchange gdp for gtp, in the absence of the 7tm-rgs, the gα subunit is gtp bound and the gβγ dimer is freed. sustained activation of g signaling involves physically uncoupling 7tm-rgs from the g protein complex to allow spontaneous nucleotide exchange and release of the gβγ dimer. the cell accomplishes this physical uncoupling by endocytosis of the 7tm-rgs whereby it cycles through the endosome while the gtp-bound gα subunit remains on the plasma membrane. it is abundantly clear that endocytosis of atrgs increases the active g protein pool. upon activation, the gtp-bound gα subunit releases the gβγ dimer enabling both g protein components to interact with cellular targets. the gα subunit is a positive modulator of cell proliferation in arabidopsis and the gβγ dimer, among other roles it plays, operates in the upr, which is important for cell survival during er stress. salt induces the upr in which case the upr alleviates nacl stress by increasing er-associated protein degradation. failure to do so triggers salt-associated-cell-death. agb plays a positive role in the upr response, since three tested loss-of-function agb mutants showed hypersensitivity to tunicamycin. the genetic data here support this biological context. in the rgs1- mutant, two important activities are increased at the plasma membrane: a proliferative factor and an er-stress reliever factor. consequently, mutations that promote active gα subunit and gβγ dimers confer sustained growth and tolerance to nacl compared to wt. consistent with this idea, disruption of the heterotrimer in a way that promotes active atgpa and agb without the loss of atrgs also conferred tolerance to nacl compared to col-. the gpa1- phenotype is also consistent with this conclusion; in the absence of atgpa plants have more activated agb at the plasma membrane as for loss of atrgs therefore these mutants are less sensitive to the stress. however, gpa mutants lack the cell proliferation function, therefore they do not behave phenotypically exactly like rgs mutants. in contrast, agb1- and agb1-2/ rgs1- double mutants lack gβ thus making these seedlings highly sensitive to the er-stress imposed by nacl. while it is possible that atrgs senses na+, we do not favor this view since the nacl effect is slow, thus activation may be indirect through an increase in glucose by regulation of glucose metabolism enzymes leading to increased sugar levels. in fact, it was clearly demonstrated that nacl increases sugar levels in root and leaf cells. this also explains the ameliorative effect of applied glucose on nacl responsiveness.  plant tolerance to nacl, in particular the recovery phase, involves the plasma membrane g protein-mediated glucose-signaling pathway. the mechanism for survival to salt stress requires g protein activation by releasing freed gα subunits and gβγ dimers. the discovery here of sodium-induced activation of g signaling via atrgs endocytosis, whether or not direct or indirect through increased glucose levels, raises further complexity involving feedback loops that will need to be addressed. 
despite being components of a singular signaling complex loss of the gβ subunit conferred accelerated senescence and aborted development in the presence of na+, whereas loss of atrgs conferred na+ tolerance evident as less attenuated shoot growth and senescence. plant growth and cell proliferation is, in part, controlled by the concerted activity of the heterotrimeric g-protein complex with glucose. less is known about pathways connecting nacl and cell proliferation and cell death. site-directed changes in the gα and gβγ protein-protein interface were made to disrupt the interaction between the gα and gβγ subunits in order to elevate free activated gα subunit and free gβγ dimer at the plasma membrane. due to long-term irrigation use in agricultural systems, soil salinity is increasing; consequently crop yield is adversely affected. prompted by the abundance of stress-related, functional annotations of genes encoding proteins that interact with core components of the arabidopsis heterotrimeric g protein complex, we tested the hypothesis that g proteins modulate plant growth under salt stress. glucose in the growth media improved the survival under salt stress in col but not in agb1- or rgs1- mutants. these results demonstrate a direct role for g-protein signaling in the plant growth response to salt stress. the contrasting phenotypes of agb1- and rgs1- mutants suggest that g-proteins balance growth and death under salt stress. the phenotypes of the loss-of-function mutations prompted the model that during salt stress, g activation promotes growth and attenuates senescence probably by releasing er stress. it is known that salt tolerance is a quantitative trait supported by genes affecting ion homeostasis, ion transport, ion compartmentalization and ion selectivity. plant growth is plastic, able to rapidly adjust to fluctuation in environmental conditions such as drought and salinity.
 plant growth is plastic, able to rapidly adjust to fluctuation in environmental conditions such as drought and salinity. due to long-term irrigation use in agricultural systems, soil salinity is increasing; consequently crop yield is adversely affected. it is known that salt tolerance is a quantitative trait supported by genes affecting ion homeostasis, ion transport, ion compartmentalization and ion selectivity. less is known about pathways connecting nacl and cell proliferation and cell death. plant growth and cell proliferation is, in part, controlled by the concerted activity of the heterotrimeric g-protein complex with glucose. prompted by the abundance of stress-related, functional annotations of genes encoding proteins that interact with core components of the arabidopsis heterotrimeric g protein complex, we tested the hypothesis that g proteins modulate plant growth under salt stress.  na+ activates g signaling as quantitated by internalization of arabidopsis regulator of g signaling protein. despite being components of a singular signaling complex loss of the gβ subunit conferred accelerated senescence and aborted development in the presence of na+, whereas loss of atrgs conferred na+ tolerance evident as less attenuated shoot growth and senescence. site-directed changes in the gα and gβγ protein-protein interface were made to disrupt the interaction between the gα and gβγ subunits in order to elevate free activated gα subunit and free gβγ dimer at the plasma membrane. these mutations conferred sodium tolerance. glucose in the growth media improved the survival under salt stress in col but not in agb1- or rgs1- mutants.  these results demonstrate a direct role for g-protein signaling in the plant growth response to salt stress. the contrasting phenotypes of agb1- and rgs1- mutants suggest that g-proteins balance growth and death under salt stress. the phenotypes of the loss-of-function mutations prompted the model that during salt stress, g activation promotes growth and attenuates senescence probably by releasing er stress. 