Heterotrimeric G protein signaling is required for epidermal cell death in rice

In rice ( Oryza sativa L.) adventitious root primordia are formed at the nodes as part of normal development. Upon submergence of rice plants, adventitious roots emerge from the nodes preceded by death of epidermal cells above the root primordia. Cell death is induced by ethylene and mediated by H 2 O 2 . Pharmacological experiments indicated that epidermal cell death was dependent on signaling through G proteins. Treatment with GTP-γ -S induced epidermal cell death, whereas GDP-β -S partially inhibited ethylene-induced cell death. The d1 mutant of rice has repressed expression of the G α subunit RGA1 of heterotrimeric G protein. In d1 plants, cell death in response to ethylene and H 2 O 2 was nearly completely abolished, indicating that signaling through G α is essential. Ethylene and H 2 O 2 were previously shown to alter gene expression in epidermal cells that undergo cell death. Transcriptional regulation was not generally affected in the d1 mutant indicating that altered gene expression is not sufficient to trigger cell death in the absence of G α . Analysis of genes encoding proteins related to G protein signaling revealed that 4 small GTPase genes, 2 GAP genes, and 1 GDI gene but not RGA1 were differentially expressed in epidermal cells above adventitious roots indicating that G α activity is regulated posttranscriptionally.


INTRODUCTION
addition, d1 displayed reduced sensitivity toward 24-epi-brassinolide in the inhibition of root growth, and coleoptile elongation in rice indicating that Gα may be linked to brassinosteroid signaling or activity (Wang et al., 2006;Oki et al., 2009).
Heterotrimeric G proteins are also involved in stress-related processes such as in oxidative stress induced by ozone (O 3 ) treatment (Joo et al., 2005). Null mutations of the single Gα and Gβ subunits of Arabidopsis showed different responses. gpa1 mutants lacking Gα were more resistant to O 3 -induced damage, whereas mutants lacking the Gβ subunit were more susceptible. O 3 is known to trigger an oxidative burst, i.e. production of high levels of hydrogen peroxide (H 2 O 2 ) similar to that observed in the biotic hypersensitive defense response (HR). WT plants showed biphasic H 2 O 2 production when treated with O 3 . gpa1 plants no longer produced elevated H 2 O 2 in the presence of O 3 . The agb1 null mutant, which is defective in the Gβ subunit, showed the late but not the early peak of H 2 O 2 production, indicating that the early phase of H 2 O 2 production required both Gα and Gβ while the later phase required only Gα (Joo et al., 2005).
G proteins are involved in the plant defense response. agg1 mutants lacking one of the two Arabidopsis Gγ subunit genes had reduced resistance to necrotrophic pathogens (Trusov et al., 2007). In rice, Gα likely participates in disease resistance.
Transcript levels of RGA1 were elevated in response to an avirulent rice blast strain. Similar induction of RGA1 was observed after application of a sphingolipid elicitor (Suharsono et al., 2002). In accord with the known fact that reactive oxygen species (ROS) act as signals in disease resistance, sphingolipid elicitor treatment caused production of H 2 O 2 in suspension cultured rice cells. d1 mutant lines with reduced GAP genes were identified (Jiang and Ramachandran, 2006). Guanine-nucleotide exchange factors (GEFs) catalyze the exchange of GDP with GTP thereby restoring the active state of G proteins (Sprang, 2001).
Flooding is a frequent environmental stress to which plants get exposed to (Bailey-Serres and Voesenek, 2008). It results in a suboptimal supply of oxygen which affects growth and development, and ultimately threatens survival of plants.
Rice is a semiaquatic plant and as such well adapted to survive hypoxic conditions.
In rice, adventitious root initials develop at the nodes of the rice stem as part of regular development (Bleecker et al., 1986). Upon submergence, adventitious roots emerge from the stem to support or replace the primary root system in the soil which becomes disfunctional at anaerobic conditions. Epidermal cells that are located at the nodes above adventitious root primordia undergo cell death prior to onset of adventitious root growth, likely to prevent mechanical damage to the growing root tip (Mergemann and Sauter, 2000). Epidermal cell death as well as adventitious root growth is controlled by ethylene. GA promotes ethylene-induced cell death while abscisic acid (ABA) acts as a strong inhibitor (Steffens and Sauter, 2005 understand how the cell death response is mediated, a possible role of G proteins in cell death signaling was analyzed using a pharmacological approach. GTP-γ-S irreversibly binds to G proteins thus locking them in a permanently activated state. Treatment of rice cv PG56 stem sections with 0.1 mM or 1 mM GTP-γ-S for 24 h resulted in a doubling of the cell death rate from 16.1% to 33.3% or 31.4%, respectively ( Figure 1A) Figure 2A). The dwarfed shoot phenotype results from reduced internodal elongation as shown for d1-1361 ( Figure 2B). While shoot growth is strongly inhibited in d1, the formation of adventitious root primordia at the nodes of the stem is not affected ( Figure 2C). Both, wt and all three d1 lines possessed an average of 17 root primordia per node.
rates were determined after 26 h and 48 h. In wt, the cell death rate rose significantly from 13% to 41% within 48 h ( Figure 3A). In d1-248 plants, the basal cell death rate was lower than in wt and submergence did not promote cell death indicating that a Gα containing G protein may be required for cell death signaling.
To further study the level at which Gα may act in cell death signaling, stem sections of wt and d1 plants were treated with 150 μ M ethephon and cell death rates were determined after 26 h ( Similar results were obtained for the allelic lines d1-1232 and d1-1361 ( Figure 3B;  Figure 3C). d1-248, d1-1232, and d1-1361 lines displayed basal cell death rates between 0.8% and 16.5%.
In these d1 lines, H 2 O 2 had no significant effect on cell death rates which did not exceed 20% after treatment with H 2 O 2 for 48 h. In conclusion, neither submergence, nor ethylene, nor hydrogen peroxide promoted epidermal cell death in the Gαdeficient d1 lines to a degree comparable to wt supporting the view that perception or transmission of these signals is dependent on a Gα-containing G protein.

Regulation of genes encoding G proteins, small GTPases, GAPs, GEFs, GDIs and GPCRs in epidermal cells above adventitious root primordia
In order to evaluate a general role of G protein signaling in cell death induction, genes encoding G proteins, small G proteins, GTPase activating proteins (GAPs), GDP exchange factors (GEFs), GDP dissociation inhibitors (GDIs), and GPCRs (G protein coupled receptors) were identified from rice and their expression was analyzed using previously described microarray data (Steffens and Sauter, 2009a these genes encoded for a G protein or for a G protein-regulating protein indicating that these are not regulated by pro-death signals at the transcriptional level. In order to analyze, if G protein genes were differentially expressed in epidermal cells above adventitious roots prior to cell death induction we analyzed about 2600 genes that were previously found to be differentially expressed in epidermal cells above adventitious root primordia as compared to other epidermal cells (Steffens and Sauter, 2009a, b). Rice has four genes encoding heterotrimeric G protein subunits, RGA1, RGB1, RGG1, and RGG2 none of which were differentially expressed in epidermal cells. The 107 small GTPases from rice can be classified into six families, Rop, Rab, Ras, Arf, Ran, and other GTPase genes (Table S1; Jiang and Ramachandran, 2006). Of these, one Rab and two Rop genes were downregulated in epidermal cells above adventitious root primordia as compared to other epidermal cells, and a GTP-binding protein synthesis factor of the family of other GTPase genes was upregulated (Table S1). The 60 known GAP genes of rice divide into the subgroups RopGAP, RabGAP, ArfGAP, RanGAP, and other GAPs (Table S2). Of these, RopGAP10 and a member of the family of other GAPs were downregulated in epidermal cells above root primordia (Table S2). One of the three GDI genes found in the rice genome, were downregulated in epidermal cells above adventitious roots (Table S3), whereas none of the five GEF genes were differentially expressed in epidermal cells above adventitous roots (Table S4). Eleven of the 13 G protein coupled receptors which were bioinformatically predicted for rice (Gookin et al., 2008) were identified in the micorarray analysis (Table S5). None of these were regulated.
Taken together, the results showed that defined G protein genes were differentially expressed in epidermal cells above root primordia prior to cell death induction possibly enabling these cells to initiate or execute cell death.

Transcriptional regulation in response to ethylene and H 2 O 2 is not dependent on Gα
We previously reported for the rice cv PG56 that nodal epidermal cells above adventitious root primordia are covered by a cuticle that has a distinct surface structure as compared to the cuticle of epidermal cell that do not cover a root. In order to find out if this morphological distinction was conserved and thus possibly relevant to cell death signaling we performed SEM studies on cv Kinmaze. Nodal epidermal surfaces were scanned in areas above adventitious roots and in areas that did not cover adventitious roots (Figure 4). Unlike what was observed in cv PG56, epidermal cells of cv Kinmaze did not display differences in epicuticular structures irrespective of their localization, indicating that it was not a hallmark of epidermal cell death fate ( Figure 4A-C). Analysis of d1-1361 plants revealed that these had the same surface structures as wild type with no morphological differences between epidermal areas above adventitious roots and other epidermal areas ( Figure 4D-F).
It was previously reported for indica rice cv PG56 that epidermal cells which undergo cell death are molecularly distinct from those that do not (Steffens and Sauter, 2009a). In order to test if differential gene expression was conserved in japonica cv Kinmaze we selected twelve genes representative of the major categories identified, i.e. signal transduction, stress response, and ethylene synthesis (Supplemental Figure S1). MT2b is an H 2 O 2 scavenger and PAP16-like a purple acid phosphatase16-like protein. CDC6 contains a FAR1 DNA-binding domain and could be a transcriptional regulator as are HOX9, MYB, ARF2, ARF3, and ANT-like. HT1like is a Ser/Thr kinase, and BBI3-3 a Bowman-Birk type Ser protease inhibitor.
Finally, ACO1 (ACC oxidase 1) and EOL1 (ethylene overproducer1-like) are involved in ethylene biosynthesis. RT-PCR results indicated that these genes were differentially expressed in epidermal cells above adventitious root primordia of cv Kinmaze as was observed for cv PG56. Next, regulation of these genes by ethylene and H 2 O 2 was analyzed. The lag phase for induction of cell death in cv Kinmaze is longer than in cv PG56. Therefore, cv Kinmaze stem sections were treated for 20 h. death, or post-induction of cell death. This finding supports the view that G protein signaling of epidermal cell death is regulated posttranslationally.
Heterotrimeric G proteins were identified as an early mediator of stress signaling (Suharsono et al., 2002;Joo et al., 2005). Arabidopsis plants deficient in the Gβ subunit of heterotrimeric G protein were more susceptible to damage by ozone and displayed higher rates of leaf cell death than plants deficient in Gα (Joo et al., 2005). By contrast, Arabidopsis plants lacking the Gβ subunit displayed greater resistance to leaf cell death triggered by tunicamycin, whereas mutants lacking the Gα subunit were as susceptible as wt (Wang et al., 2007). This difference in response may be due to the differential localization and to unique functions of the Gα and Gβ subunits. While the Gα protein was detected at about equal amounts in the plasma membrane and in the endoplasmic reticulum (ER), the Gβ protein was more abundant in the ER. Tunicamycin induces ER stress, and Gβ may specifically participate in the control of the ER stress response (Wang et al., 2007).
In the ER stress response, knock out of the Gβ subunit resulted in altered transcriptional regulation in response to tunicamycin treatment. In rice, expression levels of nearly 3000 genes differed between epidermal cells that are located above adventitious root primordia and other epidermal cells indicating that these cells possess different molecular cell identities which, in turn, may be related to their different fates (Steffens and Sauter, 2009b). Epidermal cells above root primordia undergo cell death when triggered by ethylene or H 2 O 2 , whereas other epidermal cells will not. Formation of adventitious root primordia at the nodes was not impaired in d1 plants. Despite the presence of adventitious root primordia, epidermal cells above these did not respond to pro-death signals with cell death. Interestingly, differential gene expression between the two epidermal cell types, or in response to pro-death signals was not generally altered in d1 as compared to wt as exemplified for 12 selected genes.
The ROS scavenging capacity in d1 has not been described to date.
According to this study, transcript levels of OsMT2b (Os05g0111300) were not altered in d1. Furthermore, previously published microarray data did not reveal regulation of other genes encoding for known ROS scavengers or ROS detoxifying enzymes in epidermal cells that undergo cell death (Steffens and Sauter, 2009a) supporting the view that d1 was not impaired in ROS scavenging. It is however possible that ROS scavenging activities are posttranslationally regulated. It is conceivable that residual RGA1/D1 activity in d1 was sufficient to regulate gene expression but was insufficient to promote cell death. Ethylene-induced epidermal cell death is promoted in the presence of GA (Steffens and Sauter, 2005). Near complete loss of the cell death response in d1 may be related to a strongly lowered sensitivity of this mutant toward GA (Ueguchi-Tanaka et al., 2000). It is also conceivable that the cell death promoting activity of Gα depends on brassinosteroid signaling as brassinosteroid sensitivity is also reduced in d1 (Wang et al., 2006;Oki et al., 2009). Contributions of these hormones to Gα-mediated epidermal cell death signaling will have to be resolved in future studies. It is further possible that two signaling pathways exist. One pathway leading from ethylene or H 2 O 2 to cell death is strictly dependent on Gα signaling. A second pathway which is initiated by ethylene or H 2 O 2 is independent of Gα and results in differential expression of at least some genes. The results further showed that regulation in response to pro-death signals of the subset of genes analyzed was not sufficient to trigger cell death in the absence of Gα.
In Arabidopsis seedlings, phytochrome-dependent cell death is mediated by heterotrimeric G protein. In hypocotyls of far-red (FR) grown seedlings which were subsequently exposed to white light, a heterotrimeric G protein was shown to take part in phytochrome A-mediated signaling leading to FR irradiation-preconditioned cell death (Wei et al., 2008). The gpa1 mutant showed extenuated cell death in comparison to wt, while in the Gβ mutant agb1, cell death was intensified, indicating an antagonistic role of Gα and Gβ in this cell death pathway. In addition, ROS mediated this type of cell death. Interestingly, agb1 was more sensitive to H 2 O 2 than wt seedlings, indicating that the G protein may modify the sensitivity of the seedlings to H 2 O 2 stress. Whereas heterotrimeric G proteins have been shown to regulate cell death and their diverse roles have been studied in various plant species, no information is available on the functions of those G protein genes that were found to be differentially expressed in epidermal cells above adventitious roots. Their cellular function in cell death, cell type specification, or other cellular process has yet to be elucidated. Epidermal cell death in rice is induced by ethylene and H 2 O 2 and is accompanied by transcriptional regulation in response to these pro-death signals. The work presented here identified G protein signaling through Gα (D1) as an essential step in epidermal cell death signaling. Since no genes encoding G proteins or G protein regulatory proteins were transcriptionally controlled in dying epidermal cells after treatment with ethylene or H 2 O 2 , we conclude that heterotrimeric G protein activity is regulated posttranscriptionally. While cell death rates were strongly reduced in d1, some gene regulation was still observed in response to ethylene or H 2 O 2 in d1 indicating that D1 may act downstream of transcriptional regulation.

Plant Materials and Growth Conditions
Seeds of Oryza sativa L., indica cultivar Pin Gaew 56 (PG56) were cultivated according to Sauter (1997). Internodal stem sections were prepared from 12-to 14- week-old plants. They were excised 2 cm below the third youngest node. Seeds of

RT-PCR
18-to 23-week-old rice cv Kinmaze and d1-1361 plants were used to obtain epidermal patches above adventitious roots of the second node and epidermal patches from the epidermis approximately 5 mm above the ring of adventitious root primordia. Two biological repeats were performed using tissues obtained from stem sections that were treated for 20 h with 150 µM ethephon or 0.01% (v/v) H 2 O 2 . RNA was isolated using Tri-reagent (SIGMA ALDRICH, Steinheim, Germany) according to manufacturers' instructions. cDNA was synthesized from 100 ng of total RNA with oligo dT as primer. For amplification of the cDNA fragment, the primers and cycle numbers indicated in Table S6 were used. The PCR conditions used were as follows: one cycle at 94°C for 3 min, 25 to 30 cycles with 94°C for 30 sec, 56°C to 60°C for 1 min and 72°C for 1 min followed by final extension for 5 min at 72°C. The cycle numbers and annealing temperatures were adjusted in each case to ensure specific amplification of the cDNA in the linear range. PCR products were separated by electrophoresis on a 1% (v/w) agarose gel and visualized by ethidium bromide staining.

Statistical Analysis
Statistical analysis of cell death rates were performed with Minitab (Minitab Inc., State College Pennsylvania, USA). Rates in percent were transformed with arcsine√(x/100) to obtain normal distributed data. Comparison of means was analyzed for statistical significance with an ANOVA and Tukey-test. Constant variance and normal distribution of data were verified before statistical analysis and the P value was set to

SUPPLEMENTAL MATERIAL
The following materials are available in the online version of this article.  that are not differentially expressed in epidermal cells above adventitious roots. One gene is downregulated in epidermal cells above adventitious roots as compared to other epidermal cells. Table S4. GEF (G protein exchange factor) genes (Jiang and Ramachandran, 2006) are not differentially expressed in epidermal cells above adventitious roots.