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J. Biol. Chem., Vol. 275, Issue 38, 29579-29586, September 22, 2000

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Prohibitins, Stomatins, and Plant Disease Response Genes Compose a Protein Superfamily That Controls Cell Proliferation, Ion Channel Regulation, and Death^*

Ramgopal Nadimpalli, Nasser Yalpani^§, Gurmukh S. Johal^§, and Carl R. Simmons^¶

From  Hoffmann-La Roche, Vitamins Division, Nutley, New Jersey 07110 and the ^§ Disease Resistance and ^¶ Bioinformatics Departments, Pioneer Hi-Bred International, Inc., Johnston, Iowa 50131-0552

Received for publication, March 20, 2000, and in revised form, May 28, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Prohibitins, stomatins, and a group of plant defense response genes are demonstrated to belong to a novel protein superfamily. This superfamily is bound by similar primary and secondary predicted protein structures and hydropathy profiles. A PROSITE-formatted regular expression was generated that is highly predictive for identifying members of this superfamily using PHI-BLAST. The superfamily is named PID (proliferation, ion, and death) because prohibitins are involved in proliferation and cell cycle control, stomatins are involved in ion channel regulation, and the plant defense-related genes are involved in cell death. The plant defense gene family is named HIR (hypersensitive induced reaction) because its members are associated with hypersensitive reactions involving cell death and pathogen resistance. For this study, eight novel maize genes were introduced: four closely related to prohibitins (Zm-phb1, Zm-phb2, Zm-phb3, and Zm-phb4), one to stomatins (Zm-stm1), and three to a gene implicated in plant disease responses (Zm-hir1, Zm-hir2, and Zm-hir3). The maize Zm-hir3 gene transcript is up-regulated in a disease lesion mimic mutation (Les9), supporting a role in maize defense responses. Members of this gene superfamily are involved in diverse functions, but their structural similarity suggests a conserved molecular mechanism, which we postulate to be ion channel regulation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plants frequently respond to pathogen attack with the "hypersensitive reaction" (HR),¹ a rapid localized necrosis at the site of infection that cordons off the pathogen and limits its spread (1-3). The HR cell death phenomenon bears similarities to programmed cell death or apoptosis observed in animals (3). A group of tobacco genes were identified that caused the formation of HR-like lesions on tobacco leaves when ectopically expressed from a tobacco mosaic virus expression vector (4). One of these genes, represented by a cDNA called NG1, caused both HR-like lesion formation and induced expression of -glucanase, a pathogenesis-related protein marker for defense activation. NG1 was thus interpreted to be a novel activator of the plant HR defense system (4). The predicted peptide for the cDNA NG1 was presented as 64 amino acids with no significant homology to any known proteins (4). Independently, an Arabidopsis NG1-like cDNA (gene 106) was reported to represent an mRNA induced by the plant defense activator isonicotinic acid (5). This induction was associated with systemic acquired resistance (reviewed in Ref. 6) and occurred independent of de novo protein synthesis (5).

Prohibitins are a group of highly conserved proteins that are thought to control the cell cycle, senescence, and tumor suppression (reviewed in Ref. 7). Prohibitins negatively control the cell cycle in the early G₁ phase and specifically inhibit initiation of DNA synthesis (8, 9). Prohibitin genes appear to be expressed in many tissues and organisms, but with some modulation of expression consistent with a role in the cell cycle (7, 8, 10). Mutations or deletions of prohibitin are linked to some human breast and ovarian cancers, supporting the idea that prohibitin suppresses tumors as part of its antiproliferative function involving cell cycle control (11-13). Prohibitins are also implicated in controlling senescence and aging, with which there may be a functional link to their antiproliferative function and cell cycle control (7).

Prohibitins are localized largely in the mitochondria, especially in the inner mitochondrial membrane (7) near the periphery (14). Rat and human prohibitins possess a short transmembrane helix near their N termini, which may be integrated into mitochondrial membranes (7). As mitochondrial inner membrane proteins often control ion transport and ATP production, it has been speculated that prohibitins may be involved in these processes, in particular in mitochondrial calcium efflux, which regulates ATP formation (7). Prohibitin and the prohibitin-like protein BAP37 (also called prohibitone) have also been localized to the plasma membrane of mouse lymphocytes, where they together interact with the IgM antigen receptor and may function in signaling apoptotic programmed cell death (15). Prohibitin and BAP37 interact directly with each other in animal mitochondria; and in yeast, they control replicative life span, possibly through control of mitochondrial membrane ionic potential (16).

Stomatin is an integral membrane protein found in red blood cells. In genetic disorders in which this protein is missing, a hemolytic anemia called stomatocytosis results. In stomatocytosis, the red blood cells experience high passive diffusion of univalent cations and are often overhydrated due to an abnormally high amount of intracellular sodium and low amounts of potassium. These red blood cells assume a mouth-like shape, thus stomatocytosis, from "stoma," which is Greek for mouth (17). Stomatin is thought to function as a negative regulator of univalent cation permeability. Stomatin has a single membrane-spanning region near its N terminus, with the rest of the protein thought to be cytoplasmic (18). The molecular mechanism for stomatin function is unknown, but its cytoplasmic portion has been suggested to act as a ball and chain tether that can directly plug ion channels and may also interact with the cytoskeleton (17). Northern blots detect stomatin mRNA expression in many human tissues besides red blood cells (18).

In this work, we present eight novel full-length cDNA sequences from maize, four of which are closely related to prohibitins, three to the hypersensitive response-inducing protein NG1, and one to stomatins. We demonstrate that these eight novel plant genes, along with many animal, bacterial, plant, and fungal sequences representing prohibitins, NG1-like proteins, stomatins, and other membrane proteins, compose a novel protein superfamily. Although these genes are involved in diverse physiological processes, their structural similarity suggests that they possess a related biochemical function.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Gene Isolation-- The eight full-length maize cDNAs presented in this study were identified in the EST collection at Pioneer Hi-Bred International, Inc. mRNA sources were from various tissues and treatments. The cDNA libraries were created at Pioneer Hi-Bred, and the ESTs were generated at Human Genome Sciences. The NG1 (HIR), prohibitin, and stomatin homologs were identified with the aid of the IRIS software package from Human Genome Sciences, which includes the BLAST algorithm, through which homology was indicated to tobacco NG1 (GenBank^TM/EBI Data Bank accession number U66271), to prohibitins from various species, and to human stomatin (accession number U33925). Full-length insert sequences were produced at Pioneer Hi-Bred by primer walking using an ABI 377 sequencing machine. Sequences were assembled using Sequencher^TM Version 3.0 (Gene Codes, Ann Arbor, MI) and/or AssemblyLIGN^TM (Eastman Kodak Co.) software.

Protein Sequence Analysis-- Initial public database searches were carried out using the BLASTP program (19) with a chickpea HIR-like gene (NCBI Protein Database accession number gi 3928150) as a probe, followed by PSI-BLAST (20) with default parameters (Blosum 62, gap existence cost 11, per residue gap cost 1,  ratio = 0.85, expect threshold 10). About 24 sequences that appeared as significant hits, both in terms of statistical threshold and the type, were, along with the eight maize sequences presented herein, multiply aligned by the ClustalW program with default parameters (21). The residues were reduced to a consensus sequence according to an 80% consensus generated using the CONSENSUS program of Nigel Brown (NIMR, London). To look for conserved motifs in the 32 members included in the multiple alignment, we applied the MEME algorithm, which resulted in the detection of three highly conserved motifs (26). Highly conserved residues based in part on the MEME motifs were identified to generate a PROSITE-formatted regular expression profile to perform further data base searches by the PHI-BLAST program (22). Phylogenetic analysis was carried out by using an option within ClustalW (23) to generate multiple alignments, followed by distance calculations and tree constructions with the PROTDIST and neighbor-joining program of the PHYLIP package (24). Secondary structure predictions were carried out by the DSC algorithm using multiple sequence inputs (25). Further structural analyses were carried out by hydropathy profiles using the Kyte-Doolittle method with a 19-residue sliding window.

Gene Expression Analysis-- Plant material for mRNA expression analysis was produced from the following three maize families, each with the Les9 mutation (a disease lesion mutation of maize) segregating 1:1 among the progeny: family 1 (Mo95 18-15 × sibling wild-type +/Les9; background M14/Mo20W), family 2 (Mo94S 16-35 × sibling wild-type +/Les9; background M14W23/W23r), and family 3 (Mo95 24-3 × sibling wild-type +/Les9; background M14/W23). Of the three families, only family 1 with the Mo20W background suppresses the Les9 lesion mimic phenotype.

For the Affymetrix GeneChip^® analysis, wild-type and Les9 mutant plants from all three families were used. Plants were grown in soil in the greenhouse to the V8 stage, which is when the characteristic Les9 lesions normally begin to appear. The young, upper leaf of Les9 phenotype plants that did not yet express a lesion phenotype on that leaf and corresponding tissue from wild-type sibling plants were harvested. Using duplicate equal 2-g samples representing each of these six tissues, total RNA was isolated by the TriReagent^® method according to the manufacturer's recommendations (Molecular Research Center, Inc., Cincinnati, OH). Pooled tissue from three different plants formed one sample, and the plants used for each sample were distinct. For GeneChip^® expression analysis, 1 mg of total RNA from each sample was used for poly(A)⁺ mRNA isolation by the OligoTex resin binding method according to the manufacturer's recommendations (QIAGEN Inc., Chatsworth, CA).

Protocols for preparing in vitro transcribed biotinylated cRNA probes from poly(A)⁺ mRNA for Affymetrix GeneChip^® gene expression analysis were according to the manufacturer's recommendations (Affymetrix, Santa Clara, CA) and have been described (27). In brief, 2 µg of poly(A)⁺ mRNA/sample, described above in mRNA isolations, was used for the first strand cDNA synthesis. This involved a T7-(dT)₂₄ oligonucleotide primer and reverse transcriptase SuperScript II (Life Technologies, Inc.). The second strand synthesis involved Escherichia coli DNA polymerase I (Life Technologies, Inc.). The double-stranded cDNA was then cleaned up using phenol/chloroform extraction and phase-lock gels (5 Prime  3 Prime, Inc., Boulder, CO), followed by ethanol precipitation. For the in vitro transcription to produce cRNA, biotin-11-CTP and biotin-16-UTP, in addition to all four NTPs, were used with T7 transcriptase (Ambion Inc., Austin, TX). The in vitro transcript product was cleaned up using RNeasy affinity resin columns (QIAGEN Inc.). In vitro labeled transcript yields ranged from 60 to 80 µg/sample. They were stored at 80 °C until used. The in vitro transcript products were fragmented in acetate buffer (pH 8.1) at 94 °C for 35 min prior to chip hybridization. Equal amounts (12 µg/sample) of in vitro labeled transcript were used to probe each chip overnight. The biotinylated RNA hybridizing to the chips was labeled with a streptavidin-phycoerythrin conjugate and scanned using a confocal fluorescence microscope. Expression intensity was determined as described (27). Comparisons of mRNA abundance (cRNA abundance) were made per repetition between the Les9 and wild-type samples. The average -fold change and S.E. for all the repetitions per family were calculated and are presented.

The GeneChip^® used in these experiments was constructed by Affymetrix using a set of 1501 maize cDNA ESTs, representing nearly as many genes. The genes used to produce this GeneChip^® encompass many physiological processes; perhaps one-third could be defense-related based on their homology to known or suspected defense-related genes. Two of the 1501 ESTs represented Zm-hir3 (ESTs CMSAR19R and CBPCC63R). Each cRNA GeneChip^® probing was replicated two or three times (repetitions A, B, and C). In brief, the 1.28 × 1.28-cm GeneChip^® contains a high density array of 20-mer oligonucleotides affixed to a silicon wafer. These oligonucleotides were synthesized in situ on the silicon wafer by a light-dependent combinatorial chemical synthesis (27). The oligonucleotide sequences are complementary to the sense strand of cDNA ESTs from Pioneer Hi-Bred. For each gene, there are up to 40 20-mer oligonucleotides synthesized. Twenty of these oligonucleotides are exact matches to different, although sometimes overlapping, regions of the EST. The other 20 oligonucleotides contain one base mismatch in the center, which changes hybridization efficiency. (For a minority of genes, there were <20 oligonucleotide probe pairs, but never <15 pairs per gene.) The perfect match and mismatch oligonucleotide probe pairs for each gene are tiled in adjacent regions of the GeneChip^®. Comparisons of the hybridization intensities between different perfect match oligonucleotides for a given gene and between perfect match to mismatch hybridization intensities for an oligonucleotide pair are used to determine the overall hybridization to the gene and hence its level of mRNA abundance in the samples (27).

For Northern blot analysis, tissues from wild-type and Les9 mutant plants from family 3 were used. Plants were grown in soil in the greenhouse to the V8 stage. Leaf blades (minus midribs) that had developed Les9 lesions on the mature half of the leaf tissue were harvested and divided into the basal lesion-free zone; the transition zone, where lesions were starting to form; and the leaf tip, where lesions had reached a mature stage. Corresponding tissues from wild-type siblings were also harvested. Ten micrograms of total RNA was mixed with running dye containing ethidium bromide and electrophoresed at 60 V for 15 h on 0.8% agarose gels in MOPS containing formaldehyde essentially as described (28). The gels were blotted onto nylon-backed nitrocellulose membrane and probed with a 1.35-kilobase pair insert from the CMSAR19R cDNA representing Zm-hir3.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Three distinct maize cDNAs with high homology to the tobacco NG1 peptide (GenBank^TM/EBI Data Bank accession number U66271) were identified in the EST collection at Pioneer Hi-Bred International, Inc., and their complete full-length sequences were produced. These three genes were named Zm-hir1 (accession number AF236373), Zm-hir2 (accession number AF236374), and Zm-hir3 (accession number AF236375) for Zea mays hypersensitive induced reaction genes 1, 2, and 3, respectively. Initial searches of maize hir genes using the BLAST program (19) against the public data bases indicated some similarity to prohibitins and stomatins, which prompted a search for maize cDNA clones related to prohibitins and stomatins from the same EST collection. Four distinct full-length prohibitin-like maize clones were identified and sequenced, namely Zm-phb1 (accession number AF236368), Zm-phb2 (accession number AF236369), Zm-phb3 (accession number AF236370), and Zm-phb4 (accession number AF236371). In addition, one full-length stomatin-like clone, named Zm-stm1 (accession number AF236372), was also identified and sequenced. Public data base searches did not reveal a previously reported plant stomatin-like gene. Pairwise alignments of the three maize HIR proteins showed high levels of similarity among themselves (>80% identity) and to HIR-like proteins from tobacco, chickpea, and Arabidopsis (>80% identity). Pairwise amino acid similarities of plant HIR and HIR-like proteins with maize prohibitins were between 28 and 36%, and those with maize stomatin Zm-stm1 were between 34 and 37%. This suggested that the maize HIR proteins were somewhat closer in amino acid sequence to stomatins than to prohibitins.

The non-redundant protein data base at NCBI was searched using the PSI-BLAST program (20) with a hypothetical protein from chickpea (accession number gi 3928150) as a probe, which has >90% amino acid similarity to the maize HIR proteins. This search identified many genes, including stomatins and integral membrane proteins (E < 10¹⁶), prohibitins (E < 10⁸), and HFLK/HFLC proteins (E < 10⁶). Twenty-four of these public sequences, along with the eight maize sequences introduced above, were used to generate an unrooted dendogram (Fig. 1). This dendogram revealed a large superfamily with at least four constituent families. The stomatins and integral membrane proteins, including a mechanosensor protein from Caenorhabditis elegans (accession number gi 2493263), formed a large family containing sequences from diverse phyla. A second family was composed of HIR and HIR-like sequences from plants. The family consisting of stomatins and integral membrane proteins was most closely related to the HIR family. The third family was composed of prohibitins and related sequences from diverse phyla. The bacterial membrane proteins HFLK/HFLC formed a small fourth family.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   An unrooted dendogram showing relationships among 32 members of the PID superfamily. The dendogram was generated by the neighbor-joining method using the PHYLIP 3.572 package (24). The dendogram clearly shows partitioning of families within the superfamily. The larger group (with branches colored in red) is composed of stomatins and integral membrane and hypothetical proteins that may possibly have some ion channel-regulating activity in different organisms. The family containing prohibitins is in blue. The plant HIR protein family is in green. The bacterial membrane proteins HFLC and HFLK involved in lysogenization form a separate group colored brown.

Amino acid sequences for 32 members of this superfamily were also multiply aligned to reveal shared and diverged features (Fig. 2). The coding region lengths for the HIR proteins (242-286 amino acids) are comparable to those of prohibitins (272-289 amino acids) and many of the stomatins and other membrane-associated proteins (249-481 amino acids). Relative to prohibitins and stomatins, the HIR proteins are typically shorter at the N terminus. Several regions of the protein superfamily are highly conserved and aligned well with fewer gaps. Two residues, Asp and Ala (corresponding to amino acids 64 and 167, respectively, in Zm-HIR1, being used here as a reference superfamily member), are completely conserved among all the proteins, suggesting a critical role for these residues in the biological function of these proteins. Other amino acids and structural groups of amino acids are also conserved in the PID superfamily, as shown in the consensus sequence (Fig. 2). Also depicted in Fig. 2 are the consensus DSC predicted secondary structures of each of the families within the PID superfamily. Each of the four families within the PID superfamily share secondary structural features in the same general relative positions, further indicating a relationship between these proteins.

View larger version (184K): [in this window] [in a new window]   Fig. 2.   Fig. 2. Multiple sequence alignment and consensus and DSC secondary structure predictions for the PID superfamily. A multiple alignment of 32 amino acid sequences for representative members of the PID superfamily was constructed by the ClustalW program and then manually refined: HIR proteins (green bar), stomatins and membrane proteins (red bar), HFLK proteins (brown bar), and prohibitins (blue bar). Highlighted are identical (red) or similar (blue) residues shared by at least three families within the PID superfamily. The alignment was used to generate a consensus sequence, which spans the region corresponding to amino acids 1-253 on Zm-HIR1. The consensus sequence at the bottom was based on conservation of a residue at any given position in >80% of sequences. Amino acids conserved 80% or more are shown in uppercase red letters, and two 100% conserved amino acids (Asp and Ala) are shown in uppercase and red-underlined letters. The abbreviations for amino acid structural groups are in lowercase letters as follows: o, alcohol (Ser, Thr); l, aliphatic (Ile, Leu, Val); a, aromatic (Phe, Trp, Tyr); c, charged (Asp, Glu, His, Lys, Arg); h, hydrophobic (Ala, Cys, Phe, Gly, His, Ile, Lys, Leu); , negative (Asp, Glu); p, polar (Cys, Asp, Glu, His, Lys, Asn, Gln, Arg, Ser, Thr); +, positive (His, Lys, Arg); s, small (Ala, Ser, Thr, Val); u, tiny (Ala, Gly, Ser); t, turn-like (Ala, Cys, Asp, Glu, Gly, His, Lys, Asn, Gln, Arg, Ser, Thr); and dot, any residue or gap. The location of the PID superfamily regular expression is identified by arrows. The location of the stomatin PROSITE signature is similarly indicated. Shown below the consensus sequence is the secondary structure predictions that were carried out using the latest version of the DSC algorithm (44), the prediction accuracy of which is >72%: DSC_ALL, DSC_HIR, DSC_PHB, DSC_STM, and DSC_HFLK represent consensus predictions for all the PID superfamily sequences and all the members of the respective families of HIR proteins, prohibitins, stomatins, and HFLK proteins, respectively. C, coil; E, -strands; H, helix; dot, gaps.

A systematic search for conserved motifs among the aligned sequences was performed using the MEME algorithm. The MEME motifs have been indicated as reliable indicators of family membership (26). The search resulted in the identification of three conserved motifs (Fig. 2). Using Zm-HIR1 as reference again, the amino acid positions of these motifs are as follows: motif 1, 108-167; motif 2, 56-80; and motif 3, 23-88. The relative spatial positions of these three motifs in all these genes appear to be spatially well conserved, indicating the possibility for a similar structural orientation in three-dimensional space. Motif 2 is a subset of motif 3 and is conserved in all members in the alignment. All three motifs are present in all members of the superfamily, except the HFLK/HFLC proteins, which contain only motif 2. HFLK and HFLC are bacterial membrane proteins with protease activity and are involved in lysogenization. They appear to be more distantly related to the other members of this superfamily.

Based upon the amino acid alignment and the motifs derived from the MEME algorithm, in particular MEME motif 1, we created a regular expression and used it to search public protein data bases as a pattern seed using the PHI-BLAST program (22). This PROSITE-formatted regular expression for the PID superfamily is [ILM]-[RK]-X(2)-[VLI]-[PGA]-X(10,11)-[RK]-X(2)-[VLI]-X(7)-[VLIM]-X(6)-[WFY] and corresponds to amino acids 105-139 on Zm-HIR1 (Fig. 2). Using PHI-BLAST and this regular expression, we retrieved 98 sequences that were above the threshold of 0.001 and displayed very significant E values. Of these, the HIR proteins, stomatins, and other membrane-associated proteins had E values <10⁴, and prohibitins had relatively higher E values (E = 0.003-10.0). This seed pattern was thus effective at retrieving members for each of these three families within the superfamily. In the PROSITE dictionary, the stomatin (band 7) signature has been listed as R-X(2)-]LIV]-[SAN]-X(6)-[LIV]-D-X(2)-T-X(2)-W-G-[LIV]-[KRH]-[LIV]-X-[KR]-[LIV]-E-[LIV]-[KR]. This PROSITE signature corresponds to amino acids 121-149 on Zm-HIR1, and so it partially overlaps with the PID signature. However, the PID signature accounts for all superfamily members, not just the stomatins. The C-terminal half of the stomatin PROSITE signature extends beyond the C terminus of the PID signature, and this portion is very stomatin-specific and diverged from the other PID superfamily members (Fig. 2). The PID signature partially overlaps with MEME motif 1, and although this motif 1 is not well conserved in HFLK/HFLC proteins, the PID signature nonetheless recognizes the HFLK/HFLC proteins, affirming that the HFLK/HFLC proteins are indeed distant members of this superfamily. This regular expression pattern presented herein thus represents a signature for all four families contained within this PID superfamily.

A comparison of protein hydropathy plots of maize HIR sequences with prohibitins from maize and Trypanosoma brucei revealed similar structural profiles. A similar comparison of a stomatin-like gene from Synechocystis sp. with Trypanosoma prohibitin also indicated structural similarity between several regions of these genes (Fig. 3). The shared hydropathy plots further indicate that there are conserved structural features between these diverse proteins from widely diverged phyla. Hydropathy analysis of the HFLK/HFLC proteins indicated that these genes have fewer structural similarities in common with other members in this superfamily.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Hydropathy profiles of paired members of the PID superfamily according to the Kyte-Doolittle method. Upper panel, Zm-HIR1 versus Zm-PHB4; center panel, maize Zm-HIR1 versus T. brucei prohibitin (Tb-PHB; NCBI Protein Database accession number gi 2952299); lower panel, T. brucei prohibitin versus Synechocystis sp. stomatin (Syn-STM; accession number gi 2493271). Note the close correspondence of the hydropathy profiles.

Plant disease lesion mimics are plant variants or mutants that bear symptoms of disease even though they are not infected. Such lesion mimics are common and diverse, and they are under extensive study to understand the link between plant programmed cell death and disease responses (29, 30). One such mutant of maize is Les9 (partially dominant), which is characterized by numerous spontaneous chlorotic to necrotic lesions that occur by the 9-14 leaf stage (31). The Les9 mutant also shows enhanced resistance to Bipolaris maydis and enhanced expression of defense-related proteins.² For this study, Les9 leaves of V8 plants, a stage just prior to the formation of spontaneous lesions, were investigated for altered levels of Zm-hir gene expression using the Affymetrix GeneChip^® microarray mRNA profiling technology. The Zm-hir3 cDNA was represented twice on a GeneChip^® representing 1501 genes. A small set of nearly 70 genes were observed to have a 2-fold or more change in mRNA abundance. Many of these genes are defense-related; others are unknowns or genes not generally understood to be defense-related. Among this set of 70 were the two examples of Zm-hir3. Zm-hir3 showed 2.5-8.1-fold enhanced expression in immature seedling, lesion-free leaves of Les9 mutants compared with those of wild-type siblings in families 2 and 3 (Table I). Northern hybridizations confirmed these microarray results by showing higher Zm-hir3 transcript levels in Les9 than wild-type plants in family 3 (Fig. 4). This elevated Zm-hir3 expression was detected prior to the development of a visible lesion phenotype and was at a stage when Les9 tissue shows enhanced resistance to B. maydis. When Les9 was crossed into the Mo20W background, which considerably suppresses the Les9 lesion mimic phenotype (family 1), the Zm-hir3 expression was not elevated; in fact, it was reduced 2.2-3.3-fold relative to wild-type siblings in the Mo20W background (Table I). Taken together, these results indicate that the Zm-hir3 gene exhibits modulation in mRNA expression in correspondence to the Les9 disease-related phenotypes.

View larger version (79K): [in this window] [in a new window]   Fig. 4.   Northern blot determination of Zm-hir3 mRNA abundance for family 3 wild-type and Les9 plants. Leaf blades that had developed Les9 lesions on the mature half of the leaf tissue were harvested and divided into the basal lesion-free zone (BL); the transition zone, where lesions were starting to form (TR); and the leaf tip, where lesions had reached a mature stage (TP). Corresponding tissues from wild-type siblings were also harvested. The probe was the Zm-hir3 gene (EST CMSAR19R). In the upper panel, note that the hybridization intensity of Zm-hir3 mRNA is higher in Les9 than wild-type plants for all three portions of the leaves tested. This affirms the GeneChip® result. In the lower panel is a control showing the ethidium bromide-stained gel and equality of rRNA loading per lane.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The eight novel maize gene sequences introduced in this study are structurally related to previously reported prohibitins, stomatins, and a group of plant defense-related proteins that we named HIR. By various primary and secondary structure comparisons, we have shown that these proteins all belong to a large protein superfamily present in diverse phyla. The mRNA expression pattern of the Zm-hir3 gene in the Les9 genetic background associates the Zm-hir3 gene with maize defense responses. These results are therefore compatible with the tobacco HR study (4) and the Arabidopsis systemic acquired resistance study (5), implicating HIR gene involvement in plant defense. They further suggest that a death/disease response function of these HIR genes is conserved in diverse monocot and dicot plants.

The sequence and structural similarities of plant HIR proteins to prohibitins, stomatins and other integral membrane proteins, some of which, in particular stomatins, are known to regulate ion channel function, suggest that the HIR proteins are involved in hypersensitive reaction and cell death through the regulation of ion channel activity. The C-terminal region of stomatin is very rich in -helical content and has been postulated to act as a plug to regulate potassium ion channels (17, 32). The HIR and prohibitin proteins are also predicted to have helical content near their C termini (Fig. 2), suggesting there may be a similar structure and function of this region to that of the stomatin C terminus.

Studies indicate that in animal models such as tumor cell lines, potassium plays a significant role in the maintenance of membrane potential and integrity and controls apoptosis (33) and energy conservation across membranes (34). In Drosophila, apoptotic proteins (Rpr and grim) have been shown to stably block shaker-type K⁺ channels to induce apoptosis (35). In plants, potassium is also involved in control of cellular homeostasis and cytoplasmic pH (36). In plants attacked by pathogens, there is often an efflux of cellular potassium, causing cellular acidification and extracellular alkalinization (36). Potassium levels in animals can activate several enzymes, including caspases, which are involved in apoptosis (37, 38). Although caspases or their functional equivalents are poorly understood in plants, a recent study indicated that caspase inhibitors blocked plant HR and cell death (39). In the same study, the amount of cell death was correlated to leakage of ions from leaf discs.

Tumor suppressor genes are known to regulate both cell proliferation and cell death (9, 40). Prohibitins act as negative regulators of cell proliferation in mammals and are implicated in tumor suppression (7). Some tumor suppressor genes, when overexpressed, are known to cause cell death (41, 42). The HIR genes, when overexpressed, also cause cell death (4). Given the fact that HIR proteins are structurally related to prohibitins, they may represent a novel class of plant tumor suppressors. Prohibitins have been shown to suppress the G₁-to-S phase transition in the cell cycle. If HIR proteins have a similar function, they likely also act at the G₁-to-S phase checkpoint. The retinoblastoma (Rb) and p53 tumor suppressor genes are known to act at the G₁-to-S phase checkpoint. Interestingly, suppression of K⁺ channel activity in a tumor cell line by a potassium channel blocker blocks G₁-to-S transition by keeping Rb in a dephosphorylated state (43), indicating that potassium channels function in cellular proliferation signal transduction. There is some indication that prohibitin may be involved in ion control in mitochondria (7). However, this is the first report showing a structural relationship of prohibitins to stomatins, which are known potassium channel regulators, suggesting that their common molecular function is ion channel regulation.

In conclusion, this study demonstrates that the HR-activating protein NG1 belongs to a novel gene family, which we named HIR. This family is conserved in monocot and dicot plants and appears to play a role in cell death, especially in relation to disease responses. This HIR family is, in turn, part of a large structurally related superfamily of proteins widespread in the biosphere, which includes prohibitins, stomatins, and other membrane proteins. Members of this PID superfamily are involved in cell proliferation, ion channel activity, and cell death. We postulate that these genes are generally involved in controlling ion channels, in particular potassium ion channels, and that through this control, they affect regulation of seemingly diverse processes ranging from cell division, osmotic homeostasis, and cell death.

	ACKNOWLEDGEMENTS

We thank Mary Beatty, Steve Briggs, Karen Bruce, Bill Gordon-Kamm, Eric Karrer, Keith Lowe, Pedro Navarro, John Tossberg, and Mark Whitsitt for advice and assistance.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Bioinformatics Department, Pioneer Hi-Bred International, Inc., 7250 N. W. 62nd Ave., Emerson, P. O. Box 552, Johnston, IA 50131-0552. Tel.: 515-270-5949; Fax: 515-334-4729; E-mail: simmonscr@phibred.com.

Published, JBC Papers in Press, June 21, 2000, DOI 10.1074/jbc.M002339200

² N. Yalpani, unpublished data.

	ABBREVIATIONS

The abbreviations used are: HR, hypersensitive reaction; EST, expressed sequence tag; MOPS, 4-morpholinepropanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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