The <it>Arabidopsis thaliana </it>response regulator ARR22 is a putative AHP phospho-histidine phosphatase expressed in the chalaza of developing seeds

Abstract Background The Arabidopsis response regulator 22 (ARR22) is one of two members of a recently defined novel group of two-component system (TCS) elements. TCSs are stimulus perception and response modules of prokaryotic origin, which signal by a His-to-Asp phosphorelay mechanism. In plants, TCS regulators are involved in hormone response pathways, such as those for cytokinin and ethylene. While the functions of the other TCS elements in Arabidopsis, such as histidine kinases (AHKs), histidine-containing phosphotransfer proteins (AHPs) and A-type and B-type ARRs are becoming evident, the role of ARR22 is poorly understood. Results We present evidence that ARR22 is a preferentially cytoplasmic protein, exclusively expressed in the chalaza of developing seeds. ARR22 specifically interacts with AHP2, AHP3 and AHP5 in yeast and living plant cells. Two new loss-of-function alleles, arr22-2 and arr22-3, were isolated and characterized. With respect to their morphology and metabolite status, no significant difference in the developing seeds of the arr22 mutants was observed compared to wild type. The genetic complementation of the arr22 mutants with a genomic ARR22 fragment resulted in plants (arr22/gARR22) with a pleiotropic phenotype of different penetrance. This phenotype was not observed when the phosphorylatable Asp74 of ARR22 was changed to either a dominant-active Glu or a dominant-inactive Asn. The phenotype of the arr22/gARR22 plants was comparable to that of multiple ahk, ahp and B-type arr mutants. Conclusion Our results favor the model that ARR22 acts as a phospho-histidine phosphatase on specific AHPs in the cytoplasm of Arabidopsis chalaza cells. The lack of any aberrant morphological and metabolite phenotype in the seeds of the arr22 mutants indicates that ARR22 is probably primarily responsible for the fine tuning of specific branches of chalaza-based TCS signalling. Even when slightly mis-expressed, ARR22 interferes with hormone homeostasis in non-chalaza tissues. Our data indicate that the chromatin status might play a crucial role in maintaining the chalaza-restricted expression of ARR22.</p

The response regulator ARR22 is a putative AHP phospho-histidine phosphatase expressed in the chalaza of developing seeds-4

On in black, UTR in grey), introns as thick lines, and the T-DNA insertions as triangles. The sequences at the insertion of the T-DNA (lower case letters) into the genome (upper case letters) are given for both alleles (, ). Arrows indicate the sites of primers used for RT-PCR analysis. (B) End-point RT-PCR analysis of the steady-state level of transcript. The cDNA was derived from total RNA extracted from siliques of the two allelic homozygous T-DNA insertion lines (, ) and wild type (Col-0). PCR was perfomed with the -specific primers indicated in (a) and, as a control, with -specific primers. To exclude any cross-contamination and contamination with genomic DNA, the RT-PCR was performed in the absence of total RNA or without its reverse transcription.<p><b>Copyright information:</b></p><p>Taken from "The response regulator ARR22 is a putative AHP phospho-histidine phosphatase expressed in the chalaza of developing seeds"</p><p>http://www.biomedcentral.com/1471-2229/8/77</p><p>BMC Plant Biology 2008;8():77-77.</p><p>Published online 15 Jul 2008</p><p>PMCID:PMC2478664.</p><p></p

The response regulator ARR22 is a putative AHP phospho-histidine phosphatase expressed in the chalaza of developing seeds-7

Sphorylatable Asp74 (D74) was mutated either to a Glu () or an Asn () are shown.<p><b>Copyright information:</b></p><p>Taken from "The response regulator ARR22 is a putative AHP phospho-histidine phosphatase expressed in the chalaza of developing seeds"</p><p>http://www.biomedcentral.com/1471-2229/8/77</p><p>BMC Plant Biology 2008;8():77-77.</p><p>Published online 15 Jul 2008</p><p>PMCID:PMC2478664.</p><p></p

The response regulator ARR22 is a putative AHP phospho-histidine phosphatase expressed in the chalaza of developing seeds-9

Ive RT-PCR. Data are expressed as mean +/- SD (n = 3) (B) Scheme of seed anatomy adapted according to Debeaujon and colleagues []. Note the location of the chalaza. (C) Confocal images of transgenic seeds at different developmental stages expressing a construct. Bright field, GFP images and overlays are presented from the left to the right. The bars represent 100 μm. (D) Immunolocalization of GFP on cryosections prepared from developing seeds of transgenic plants using a GFP-specific antibody (anti-GFP). The nuclei of the cells were visualised by DAPI staining (DAPI). The dotted line confines the chalaza area. The bars represent 20 μm.<p><b>Copyright information:</b></p><p>Taken from "The response regulator ARR22 is a putative AHP phospho-histidine phosphatase expressed in the chalaza of developing seeds"</p><p>http://www.biomedcentral.com/1471-2229/8/77</p><p>BMC Plant Biology 2008;8():77-77.</p><p>Published online 15 Jul 2008</p><p>PMCID:PMC2478664.</p><p></p

The response regulator ARR22 is a putative AHP phospho-histidine phosphatase expressed in the chalaza of developing seeds-3

column). The right column shows the corresponding bright field images of the transformed cells. The bars represent 25 μm. (B) Western blot analysis of protein extracts derived from transiently transformed tobacco leaves assayed for BiFC fluorescence before extraction (1–6). Immunodetection of the YFP-N fusion proteins (AHPs) was carried out with an antibody against c-myc-tag (anti c-myc) and of the YFP-C fusion protein (ARR22) with an antibody against the HA-tag (anti HA). M, protein marker.<p><b>Copyright information:</b></p><p>Taken from "The response regulator ARR22 is a putative AHP phospho-histidine phosphatase expressed in the chalaza of developing seeds"</p><p>http://www.biomedcentral.com/1471-2229/8/77</p><p>BMC Plant Biology 2008;8():77-77.</p><p>Published online 15 Jul 2008</p><p>PMCID:PMC2478664.</p><p></p

The response regulator ARR22 is a putative AHP phospho-histidine phosphatase expressed in the chalaza of developing seeds-6

Ant and wild type (grey bars) by HPLC analysis as described in Methods. The data are expressed as mean ± SD (n = 5).<p><b>Copyright information:</b></p><p>Taken from "The response regulator ARR22 is a putative AHP phospho-histidine phosphatase expressed in the chalaza of developing seeds"</p><p>http://www.biomedcentral.com/1471-2229/8/77</p><p>BMC Plant Biology 2008;8():77-77.</p><p>Published online 15 Jul 2008</p><p>PMCID:PMC2478664.</p><p></p

The response regulator ARR22 is a putative AHP phospho-histidine phosphatase expressed in the chalaza of developing seeds-0

Elay (drawn lines), which converges at the canonical AHPs (AHP1 to AHP5). Subsequently, the phosphoryl residues (P) are relayed to the A-type and B-type response regulator (ARRs), which are activiated by this phosphorylation and initiate the cellular responses (broken lines with arrow heads). Phosphorelay reactions catalyzed by receiver domains enable the phosphate flow in both directions. The phosphohistidine phosphatase ARR22 efficiently de-phosphorylates AHP2, 3 and 5, releases phosphate (P) and, therefore, acts a "sink" for phosphoryl residues. Especially in tissues, in which ARR22 is normally not expressed, this "sink" action strongly disturbs the phosphoload of the TCS network and interferes with TCS-regulated developmental processes (broken lines with vertical end line) causing dramatic phenotypic abnormalities. A similar "sink" function is reported for AHK4/CRE1 in the absence of cytokinin []. The pseudophosphotransfer protein AHP6 lacks the conserved His residue essential for phosphorelays and is proposed to inhibit the phosphate flow within the TCS network by competing with other AHPs for interactions with AHKs and ARRs [].<p><b>Copyright information:</b></p><p>Taken from "The response regulator ARR22 is a putative AHP phospho-histidine phosphatase expressed in the chalaza of developing seeds"</p><p>http://www.biomedcentral.com/1471-2229/8/77</p><p>BMC Plant Biology 2008;8():77-77.</p><p>Published online 15 Jul 2008</p><p>PMCID:PMC2478664.</p><p></p