Indian Journal of Experimental Biology Vol. 39, March 200 1, pp. 19 1-200

Review Article

Germins and germjn like proteins: An overview

Debasis Patnaik & Paramjit Khurana*

Department of Pl ant Molecul ar Biology, Uni versity o f Delhi South Campus, New Delhi 11002 1, Indi a. Fax: 9 1-011 -6885270. Email : paramjitkhurana@ hotmail.com

Molecul ar investi gations during wheat germination have revealed unique developmentally regulated proteins, designated as germin s, which show remarkable resistance to broad specifici ty proteases and to dissociati on in SDS. Germins in cereals have an oxalate oxidase acti vity, which generates H20 2 from the oxidati ve breakdown of oxalate thereby pl aying a significant ro le in plant development and defense. Germin like proteins (GLPs) exhibit seq uence and structural simil ari ty with the cereal germi ns but mostly lack oxalate oxidase activi ty. Germins and germi n like proteins (GLPs) are a cl ass of developmentally regul ated glycoproteins characteri zed by a l3-barrel core structu re, a signal peptide, and are associated wi th the cell wall. GLPs exhibit a broad range of di versi ty in their occurrence and acti vi ty in organisms ranging from myxomycetes, bryophytes, pteridophytes, gymnosperms and angiosperms. Germins and GLPs are thought to pl ay a significant role during zygotic and somatic embryogenesis (wheat and Pinus, respecti vely), salt stress (barley and Mesembryanthemum crystallinum ), pathogen elicitation (wheat and barley), and heavy metal stress, etc. Characterization and cloning of some of the genes encoding germins and GLPs has facilitated a better understanding of their regulation and raised their potential of biotechno logical application.

Seed storage proteins have been extensively studied in cereals and legumes due to hi storical significance, abundance, nutrition, economic importance and more recently due to their potential biotechnological applications. These proteins are intricately associ ated with germination to meet the increasing demands of the pl ant. The search for molecular markers associated with germination revealed a protein synthesized within hours after wheat seeds were imbibed in water and named as germinl . During germination of wheat seeds, water imbibition occurs in three different phases ; in the first phase (ca. 1h), the water content of the dry wheat embryo increases from 5% to 60%, followed by a secondary lag phase (ca. 4h), and subsequently, growth and uptake of water results in 85% water content2

• Simultaneously, a widespread quantitati ve change in the translatable mRNA population takes pl ace. The first phase of water absorption leads to di sappearance of two mRNAs that encode a methionine labeled storage protein (Em protein) and a cysteine labeled Zn­metall othionein protein (Ec protei n). The secondary uptake of water leads to the synthesi s of nascent mRNA resulting in a re latively rare soluble protein. Thi s prote in which appears to be the only gene

*Correspondent author

product synthesized de novo in wheat embryos during germination , is a homopentameric glycoprotei n des ignated as germin2

. Germin acts as a signal fo r the onset of growth in germinating wheat seeds. Although several isoforms of germins exist in different cereals, not all of them are associated with germinati on3

.

Germin is a homopentameric protein whi ch does not di ssociate in di ssociating gels. and is extremely refractory to digestion by broad-specific ity proteases. This extreme stability of germins at room temperatures was utili zed to establish its amino ac id composition, CNBr fragmentation analysis and NHr terminal eicosapeptide sequence4 and was in valuable for screening and identi fying a virtual full -length cDNA clone by differenti al hybridizations. Germi ns have oxalate oxidase acti vity and are encoded by a multigene family which maps to chromosome 4A (-5 copies), 4B (-3 copies) and 40 (-9 copies) in hexaploid wheat6.

Germins are simil ar to prions In being glycosylated, stable at extremes of pH and hi ghly res istant to proteases2

• The first proteins to have any significant sequence similarity to the wheat germi n were the spherulins, expressed in the s lime mould Physarum polycephalum during pl asmodial encystment or spherul ation 7. At the amino ac id level there is 44% simil arity between the germin gf-2.8 and

192 INDIAN J EXP BIOL, MARCH 2001

spherulin 2b sequence, and reaches 60% for the centra l core sequence6

. Although the spherulins do not show oxalate oxidase activity and their bio­chemica l properties remai n to be elucidated , together wi th germins they are assoc iated with the cell walls and with phenomenon (spherul ation and germination) that relate with the water economy of the cells (desiccation and hydration). These unusual properties of germins have been explained by the reali zation that wheat germins and its relatives from other cereals are members of the "cupin" gene famill and their resis tance to extremes of environment is likely to be a fu nction of their structural similarity to other desiccation tolerant proteins including the seed storage proteins and the resistance to H20 l is due to its enzymatic generation.

Germin like proteins (GLPs) are a class of proteins which show signi ficant sequence identity (average 50%) with wheat germins, and together with germins belong to a superfamily of funct ionall y di verse pro­karyotic and eukaryotic proteins designated as cupins (from the latin term "cupa" fo r a small barreI9

.1O

.

Germins and GLPs share several interesting features; firstly, they are all glycosyl ated, but the site of glycosylation may vary within the oligomer, secondly, they are usually fo und associated with cell wall ; the presence of a signal peptide supports the apoplastic localization. Finally, and most significantly, they share a ~-barre l core structure. All the GLPs share atleast 31 % identity at the amino acid level. The most hi ghly conserved region occurs near the midd le of the protei n.

Occurrence & Distribution

Germins and GLPs are found in organisms ranging from Physarum polycephalum (s lime mould), Barbula unguiculata (a moss), Pinlls caribaea, Pinus radiata (gymnosperms), Mesembryanthemum crystal­Iinu/ll , Nicotiana sp., Prullus persica, Pisum sativul11, Fragaria amanisa, Brassica napus, Sinapis alba, Arabidopsis thaliana (dicots) and Saccharum officinale, Oryza sati va, Hordeum vulgare and Triticum aestivum (cereal monocotyledons) (see Table I). Based on the sequence similarities as well as biological characteristics of germi n like proteins, more significantly their spatial and temporal express ion pattern , Carter et al. 14 categorized GLPs into five subfamilies comprising of GLP7 (AtGLP7), gennin (TaGermin2.8, TaGermin3.8, Hv72) , GLP subfamily 1 (McGLP, AtGLP9, AtGLP6, AtGLP2a,

AtGLP2b), GLP subfamily 2 (AtGLP4, AtGLP8, AtGLP 10, AtGLP5) and the GLP su bfamil y 3 (BnGLP, PnGLP, AtGLP I, SaGLP, AtGLP3a AtGLP3b)14. The regulation of germin and GLPs is governed by developmental and environ mental cues. The accumulation pattern often di splays characteristic organ speci ficity in response to a specific developmental stage. Most GLPs are yet to be associated with an enzymatic acti vity. However, GLPs with oxalate oxidase ac tivity are reported from pea roots (genebank accession CAB65369, CAB65370, CAB65371 ) and Mn-dependent super­ox ide di smutase activity has been reported for a GLP isolated from a moss II .

Germin and oxalate oxidase activity

Oxalates are generally considered as inert end products of plant and animal metabolism. The enzyme oxalate ox idase degrades oxalate (C ZHl 0 4) to COl and HlOl in presence of oxygen. The first report of an enzyme with oxa late oxidase aclivi ty was from powdered wheat grains24. Subsequent ly, circum­stantial evidence from two independent studies25

.l6

showed sequence similarity and accumulation pattern of barley oxalate ox idase and germin isofonn led to definitive evidence that the germin isofonn made during wheat germination is an oxalate ox idase. Further characterization and cloning of germin and GLP encoding genes in wheat and barley has resu lted in the accumulation of information necessary for the understanding of the function and regu lation of thi s I f . . I 1115-17 Th . I c ass 0 protell1s 111 p ants--'- - . e commerc ia

significance of this enzyme is due to its use in di agnostic kits to assay oxalate levels in blood plasma and urine for the control of hyperoxaluri a, a condition that commonly leads to the formation of kidney and bladder stones or due to a rare genetic di sorder causing the deposition of calcium oxa late crystals throughout the bod/.

Role in de velopment

The oxalate oxidase activity and deve lopmental regulation of germins, suggest that oxa late is used in the developmental process of hi gher plants. Wheat germin is locali zed in cell walls of embryos and I-hOl is released by peroxidases in the ox ida tive cross linking of cell wall polymers28. Two features of wheat germjns are linked to specific ro le in plant development. Firstly, the association of germins to glucuronogalactoarabinoxylans (CGAX) suggests

\

PATNAIK & KHURANA: GERMINS & GERMIN LIKE PROTEINS 193

their involvement in incorporating these compounds into growing walls. Secondly, the generation of H20 2

is utilized for peroxidase mediated reactions like lignification and cross-linking of coumarates, extensins and ferulates to cell wall hemicellulases and pectins. Therefore, germins are postulated to have a role in both initiation and termination of wall expansion especially during during mid-maturation stage of embryogeny and post-germinative embryo growth?

Spatial pattern of oxalate oxidase gene expression during wheat germination2~ has revealed more enzymatic activity in roots in comparison to the coleoptiles. Expansion and elongation of cells in the root and shoot primordia result in the protrusion of the radicle from the enveloping tissues. The first detection of germin like oxalate oxidase activity occurs in the enveloping tissues (the coleorhiza, and the fused testa and pericarp of the caryopsis). The local generation of H20 2 is then utilized in cross­linking of the coleorhiza cell wall components and thereby preventing its further elaboration. The accumulation of germin like oxalate oxidase mRNA, protein and enzyme activity are localized in cell types which are characteristically restricted in the extent of cellular growth and in which peroxide-mediated crosslinking is expected to function in terminal cellular differentiation.

Germin like proteins are also found to be associated during specific developmental stages like somatic and zygotic embryogenesis, floral induction , fruit ripening, seed and wood development. Additionally, these proteins are also linked to specific plant microbe responses like nodulations in legumes

d h · 1U an pat ogel11c responses .

GLPs and auxins

Characterization of the wheat germin gf-2.8 gene and functional analysis of its promoter showed the presence of numerous putative auxin-responsive I . I 63U TI .. f h' e ements 111 t 1e promoter ' . 1e transcnptlon 0 t IS

gene was found to be stimulated by auxins, 2,4-D being the most active among the molecules tested3u

.

Studies of genes encoding auxin binding proteins (ABP) in peach has shown their significant sequence similarity with germin like proteins. ABP 19/20 of peach also has an N-terminal hydrophobic signal sequence and a putative N-glycosylation site like the GLPS31

• The deduced amino acid sequence of Alger3

from Arabidopsis shares 67% sequence similarity with the coding region of ABP20 of peach31

indicating that among the Arabidopsis GLPs, AtGER3 is related to ABP2032

. The recent catagorization of germins and GLPs along with ABPs in the class of single domain cupins has substantiated

I f . 10

the relatedness between these two c ass 0 protell1s .

Germin like proteins and somatic embryogenesis The role of germin like proteins in somatic

embryogenesis was first highlighted by Domon el at. 12 with the identification of three extracellular proteins secreted by embryogenic tissues of Pillus caribaea var. hondurensis showing highly specific serological affinity to antibodies directed agai nst wheat germin monomer. These proteins were also found to crossreact with an antiserum raised agai nst the glycosylated pentameric germin-like protein of barley. The extracellular protein GPIII present in the five embryogenic lines show strong homology with germin sequences at the nucleotide and amino acid level. Serological studies indicated that GP Ill , GP103 and GP94 are strict representati ves of embryogenic lines. The presence of germin-like proteins (GLPs), bound ionically to the walls of preglobular somatic embryos of Pinus caribaea var. hondurensis and not to nonembryogenic callus makes these proteins first markers of somatic embryo­genesis.

The physiological implication of such markers during early embryo development was revealed with the isolation of a cDNA clone, PcGERJ from somatic embryos and predicted to encode a protei n with sequences similar to GLPs 13. PcGER1, a full length cDNA of a GLP gene, expressed in somatic as well as in zygotic embryos of carribean pine, encodes a protein that possess all the features of germins and GLPs. PcGERJ transcripts were detected in all the embryogenic lines and not detected in nonembryo­genic lines. The transcripts were also present in quiescent zygotic embryos but absent in the female gametophyte, the female storage tissue of conifers. The regulation of PcGERl gene is drastically different from that of wheat gf-2.8 gene, as the transcript level of PcGERJ decreases sharply during germination. GLPs in conifer somatic embryogenes is are assumed to have a role in the initiation and termination of wall expansion, as these two processes occur when the embryogenic tissues are mai ntai ned on a proliferation medium.

194 INDIAN J EXP BTOL, MARCH 2001

Table I-Germin like proteins and coding elements from different groups of pl ants

Plant species

Fungi Physartllll polycephallllll

Bryophytes

Barbllia lInguiculata

Gymnosperms

PillllS caribaea P. radiata

Dicotyledons

Arabidopsis

Brassica naplls

Glycine 1I1ax

GOSSypill1ll hirslItlllll

Lycopersicoll esclllentlllli

Nicotiana sp.

Pharbitis lIil

PiSlI1I1 sativlIlII

Sillap is alba

Solall1l1l1 tliberoslllll

Source ti ssue

Spherules Spherulins I a and I b

Cell lines (B uGLP with Mn-SOD acti vity)

Somatic embryos Somatic embryos

Etiolated seeds Whole seedlings Green shoots (GLP I) Immature si liques (GLP2a1b) Seedling hypocotyls (GLP3b) Seedling hypocotyls, 3d (GLP5) Mixed ti ssues (o ther GLPs)

Etiolated seed ling (BnGLP)

Immature seed coats Roots Cotyledons Whole seed lings

2-3 week old leaves Mature fl owers

Immature fibre (6 dpa)

25-40d callus 8-week-old shoot meristem 4-week-old leaf

6

Reference/Accession No.

II

12 13

AA220904, AA220905 AA22089 I , AA220890 AF049065

14

U2 1743

AW307501 AW233901, AI855478 AW202 170 AI965984

AI960418,AI856484 AI930844

AI728954

AW035078 AI482753 AJ484262

Adaptation to Mn deficiency (Mdip I) ABO 12 138

Nectary ti ssues (Nectarin J)

Leaves (PnGLP I)

Stem (4d seedling) Root (ger l , ger2a, ger2b)

Leaves (SaGLP)

Tuber

15

16

AJ222979 CAB65369,

CAB6537 1

17

18

CAB65370,

(Coll td)

,

I

PATNAIK & KHURANA: GERMfNS & GERMfN LIKE PROTEINS 195

Table I-Germi n Like Proteins and Coding Elements from Different Groups of Plants-Contd

Plant species Source tissue Reference/Accession No .

Monocotyledons

Horde/III/ vlI /gare Leaf mesophyll (pHvOxOa, pHvOxOb')

Leaf(HvGLPI)

19 20

Oryza sativa Roots (RGLPI,RGLP2) AF141879, AFI41880 Shoots (GER I, GER4, GER6, GER8) AF049065

Immature seed (GLP16) Seedling (GLP II 0)

AF042489 AF141879, AF032974, AF051156 AF032974 Panicle at ripening state (GER3)

Panicle and flowering state (GER7J 21

SaccharulIl sp. Leaf ro ll AA525686

Triticlllll aestivlIlIl Germinating embryo (Gf-2.8, gf -3.8) TaGLPI , TaGLP2, TaGLP3, TaGLP4 Leaf

22 Y09915, Y09916, Y09917 , Y099 18 23

(TaGLP2a" TaGLP2b) AJ237942 , AJ237943

GLPs and clock regulation The transcription of germin like proteins has been

found to exhibit circadian oscillations in plants ranging from the long day plant Sinapis aLba (SaGLp)17 to short day plant Pharbilis niL (PnGLp)16. The GLPs exhibiting diurnal variations are leaf specific and have different courses of accumulation reflecting on the photoperiodic responses of each plant. The barley HvGLP 1 transcript also exhibits diurnal variations, the minimum and maximum of RNA abundance being at the end of the light and dark period, respectivel/o. These GLPs share significant sequence similarities amongst themselves rather than with the other GLPs from different plants involved in other phenomenon. In Arabidopsis also the Alger3 promoter, which mediates organ specific expression, displays preferential expression at the beginning of night32

. Genetic transformation with chimeric promoter-reporter gene constructs has revealed the transcriptional regulation of the Atger3 oscillations. The Alger3 promoter was found to mediate the cycling of the gus gene in a similar organ specific manner with the highest level of expression at the beginning of night. In situ hybridization experiments has indicated similarities of Atger3 expression pattern with the spatial expression of its mustard counterpart SaGLP. Characterization of the Alger3 genomic locus by deletion analysis has revealed that the clock response clements contributing to high amplitude Alger3 oscillations largely reside between -299 and

-96732. Further studies involving introducti on of antisense constructs in pl ants and their ectopic expression will aid in elucidation of the role and function of this class of developmentally regulated protein in plants.

RoLe in salt stress A correlation of germins and salt stress was

revealed with the finding that germin like proteins and coding elements undergo a change when salt tolerant monocotyledon (barley) and dicotyledon (ice plant) are subjected to salt stress33

. Synthesis of germins in roots of barley seedlings, increases transiently during salt shock and accumulates when seedlings are grown on nutrient solution containing sale4

. Hurkman and coworkers have identifi ed two developmentally regulated soluble polypeptides Gs I and Gs2 of 26 and 27 kD, respectively, in response to salt stress. The polypeptides Gs 1 and Gs2 accumulate differentially in an organ specific manner in the root and shoot, respectively. Interestingly, the levels of the 27kD polypeptide, that is serologically related to wheat germin, decreases in roots in response to salt stress. A root specific transcript in the halophyte Mesembryanthemum crystallinum also decreases during salt treatment , and shares signi ficant homology with germins33

. Germin gene expression follows a distinct spatial distribution in response to salt stress. In barley, germin expression levels were higher in roots than the vascular transi ti on region,

196 INDIAN J EXP BIOL, MARCH 2001

whereas, in wheat, the level s are highest in the vasc ul ar transition region and higher in the shoots than in the roots. In barley, the developmental regul ation of germin gene expression at the transcripti onal level is also influenced by salt stress35

,

while in wheat, it does not have a significant impact on germin abundance and oxalate oxidase activit/6

.

Role in plant defense

A possible role of germins in plant defense was suggested even before the identifi cation of its enzymatic activity. Gcrmins are conspicuously present in rust-resistant, domesticated wheat and other cereal crops37. The perox ide generating oxalate ox idase acti vity of germins is indicati ve of its signi ficant role in plant defense in response to pathogens. The generation of H20 2 is thought to have a direct antimicrobial effect by formation of active oxygen species (ox idative burst) , and it can also act as a signal fo r the action of other defense mechanisms. Perox idase mediated oxidative cross­linking of cell wall structural proteins has been implicated in increased wall resistance to fungal wall degrading enzymes by processes like li gnification26.28

.

Recently, several groups have independently reported a pathogen induced increased activity of both germin and oxalate oxidase in wheat and barle/5

.35

.38.

Germins respond to pathogen invasion as well as a number of hormonal and environmental signal s. Their expression is also found to be modulated by IAA, ABA, SA, MeSA and MeJA35

.

In Hordeum vulgare (barley), characterization of the pathogen responsive oxalate oxidase revealed minor differences fro m the commercial preparation, as well as from a barley root oxalate oxidase38

. The oxalate oxidase activity locali zes in the epidermal cells of the mature region of primary root and coleorhiza of 3d-old barl ey seedlings and in the coleorhi za alone of lOd-old seedlings. This oxalate ox idase activity was shown to be induced in barley leaves spec ifically in response to infection by the fungus Erysiphe graminis but not by wounding25

. The ex istence of two oxalate ox idase genes in the barley genome was confirmed with the isolation of two cDNAs; one pHvOxOa representing the multicopy gene encoding the major enzyme, and the other pHvOxOb' representing the single copy gene encodi ng a closely related enzyme. Both these genes accumulate transcripts in response to powdery mildew attack with pHvOxOa accumulating six times

more than the level of pHvOxOb. This oxalate oxidase (pHvOxOa) is found exclusively in leaf mesophyll tissue in association with the cell wall. These evidences indicate a possible ro le of oxalate oxidase for the regulation of hypersensitive response 19

• In a separate investigation reported the di sappearance of two extracellular protei ns of ca. 22 and 23 kD from barley leaves upon stress treatments that enhance H20 2 levels thereby induci ng resis tance to powdery mildew infection20. A 22 kD protei n was found to belong to the family of GLPs and des ignated as HvGLPl. Charac terization of thi s protein revealed a lack of oxalate oxidase activity but its transcript levels were found to be regulated by a circadian clock. 23 kD HvGLP is bound to the cell wall s by non-covalent linkages as it can be resolubili zed from the cell walls by heat or H20 2 treated leaves, or by boiling in SDS. These evidences indicate the ex istence of atleast two groups of related proteins in the same plant species2o.

Induction of germin gene expression in TriticlIlII aestivum (wheat) has been reported to be an indicator of powdery mildew infection as it is induced in both the susceptible and relatively resistant culti vars. Immunoblot and ac tivity blot analysis has demons­trated an increase in oxalate oxidase activity parallel with the accumulation of the germin oiigomer35

. In an effort to study and explore the poss ible uses of germins in plant defense the regul ati on of a gennin gene was studied in a heterologous system, tobacco. Tobacco proved to be a suitable hetero logous system as it lacks endogenous oxalate oxidase ac tivity, and the biochemical properties of the introduced transgene are conserved alongwith its hormona l and developmental specificities. The auxin responsive gJ-2.8 promoter leads to an increase of OxO activity in wheat and transgenic tobacco in response to treatment with auxins30. The express ion of germin gene was also found to be stimulated by heavy metal ions notably Cd2

+, Cu2+, C02

+ and polyamines, wou nding and TMV. In all cases increase in genn1l1 accumulation parallels with an increase in oxalate oxidase activity. The organ specific transcript accumulation is more prominent in leaves as compared with other organs. Studies employing the promoter of the wheat germin gJ-2.8 gene has revealed its insensitivity to signal s like salicylic acid, jasmonic acid, nicotinic acid and benzothi adiazole. Polyamines like putrescine and spermidi ne have been found to be activators of germjn gene express ion36.

,-

1

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PATNAIK & KHURANA: GERMTNS & GERMIN LIKE PROTEINS 197

These findings indicate the existence of more than one type of signaling pathways for regulation of germins, firstly, for development and possibly for auxin regulation, and secondly, for stress response. The regulation of this gene in wheat and transgenic tobacco thus suggests the existence of different signal transduction pathways for abiotic stress in wheae6

.

The significant role of germins and related proteins in phenomenon like plant defense and development has led to study of this class of proteins and coding elements in the model system Arabidopsis. Although Arabidopsis GLPs are not yet known to be associated with an enzyme activity that can be linked to plant defenses, GLPs are regulated differentially in different organs; AtGER2, resembles more closely the wheat germins which is found exclusively in developing embryos, AIGERl is expressed during zygotic embryogenesis, and the AtGER3 transcript is found in leaves and flowers and vary during the circadian cycle39

. Carter el al. 14, have characterized

the Arabidopsis GLP gene families and found it to consist of atleast 12 GLP genes. In Arabidopsis, with the exception of GLP4 that lacks a N-terminal signal sequence, accumulation of GLPs occurs at three different sites; in the vacuole, at the plasma membrane, and outside the cell '4 . The GLPs thus share si milar pattern of intracellular localization with that of pathogenesis related proteins in plants40

.

Genetic transformation of plants with genes encoding for germins and GLPs

Fungal pathogens of crop plants cause extensive economic damage to the farmers by reducing the yield. One of the strategies for improving fungal resistance is to strengthen the plant defense by the introduction of transgenes encoding products that inhibit the pathogenesis process. Generally the transgenes for fungal resistance are targeted towards reinforcement of plant cell walls, accumulation of phytoalexins, synthesis of ribosome-inactivating proteins, antimicrobial peptides and other patho­genesis related proteins. The extracellular localization of germins and GLPs as well as the peroxide generating oxalate oxidase activity of germins indicated a key role of this class of proteins in response to fungal pathogens. Peroxide generating cell wall oxidases like glucose oxidase41 and ascorbate oxidase42 also playa key role in cell wall reorganizat ion . Oxalic acid production by phyto­pathogenic fungi is one of the key features in the

pathogenesis process leading to the rotting of the tissue43

• Oxalic acid suppresses host plant defenses by inhibiting o-diphenol activit/4 and reduces the internal pH of the plants causing stimulated activity of cell wall degrading enzymes produced by the

4-pathogens). The mutant strains of the pathogen Sclerotinia sclerotiorum deficient in oxalate production are also avirulent46

• Due to the significant roles of oxalate in the pathogenesis process, biologists have considered genetically transforming plants with an oxalate-catabolizing gene to provide increased level of resistance against oxalate producing pathogens. Agrobacterillm-mediated trans­formation of barley oxalate oxidase gene into oilseed rape has shown that the gene encodes an active form of the enzyme, which subsequently gives tolerance to exogenously added oxalic acid43

. These studies have raised the possibility of manipulating the oxalate metabolism of plants by introducing exogenous genes thereby imparting disease resistance in plants. The expression patterns of genes encoding germins and GLPs in response to fungal pathogens has indicated the possibility of employing t!lese genes for the generation of transgenics with better fungal tole­ranee 19,20-25,30,36,38, Transient expression of the

pathogen induced germin gj-2.8 but not the HvGLP 1, that insolubilizes in response to powdery mildew infection, has resulted in the reduction of penetration efficiency of powdery mildew fungus on transformed wheat cells23. More interestingly, transient express ion of two modified wheat genes, gj-2.8 and TaGLP2a, which encode proteins that lack oxalate oxidase activity, also reduce the penetration efficiency of the pathogenic fungus . The transgene product of wheat germin gj-2.8 gene was found to insolubilze at sites of attempted fungal production where localized production of H20 2 takes place. These findings highlight the structural role of germin related proteins in cell wall reinforcement during pathogen invasion. The genes encoding germins and GLPs are thus a new addition to the class of transgenes with antifungal activity for obtaining fungal tolerance in crop plants.

Evolutionary correlation

Modern day seed storage proteins can be traced back to the spherulin like proteins of myxomycetes, which are thought to be involved in basic cellular desiccation and hydration process including osmotic regulation

47• Spherulins are putative cell wall proteins

of the slime mold Physarum polycephalum that

198 INDIAN J EXP BIOL, MARCH 2001

increase during spherul ation, a process brought on by va ri ous environmen tal stresses including drought6

.

The amino acid sequences of the seed storage globulin proteins legumin and vici lin which are synthesized and accumulated during the seed matu ration phases of both angiosperms and gymnosperms, share stati sti call y signi fica nt simi larity (up to 60% fo r the central core sequence) to the germinat ion specific germins of wheat as well as to the spherulins of myxomycetes4S

. This was evidenced by the intron-exon deri ved structu re of a spherulin gene. A vicilin like gene expressed in the cycad Zalll ia JurJuraceae revealed signi fica nt simi larity to a sucrose hi nding protein iso lated from soybean47. Conservation of ami no acid pattern in a sucrose binding protein with vici li ns of distantly re lated species along with the precise conservati on of all five int ron pos itions indicates evolutionary relationship between the polypeptides and thei r respective genes. These evidences suggest the ex istence of a superfamily of related genes including both vicilin­like and legu min-li ke seed globu lin genes as well as genes encoding for spherulins, germins and sucrose bind ing proteins . Recent isolation and characteri­zation of germin like protein (BuGLP) with Mn­superoxide dismutase act ivi ty from the moss Barbula unguiculara revea led strong sequence similarity to germin (oxa late ox idase) and GLPs of several plant species and possesses all the characteri stic features of the germi n famil y". BuGLP was the first germin like protein that was demonstrated to be a metall oprotein with Mn-SOD ac ti vi ty but no oxalase ox idase activi ty with an extracellular location. Germins and GLPs which belong to the cupin superfamil / have been recentl y placed in the class of single domain cupins based on the presence of a single conserved domain at the core of the proteins, alongwith phosphomannose isomerases (pmi), polyketide synthase, dioxygenases, spherul ins, auxi n binding protei ns (ABPs) and epi merases. This classi ficat ion has fu rther substan­tiated the evolutionary conservation of this class of proteins in organisms as diverse as microbes to hi gher pl ants and animal s.

COJlcluding remarks

The study of germins wh ich was initiated with the objective of a better understanding of the process of germination has resulted in the identification of a significant class of developmentally regul ated proteins widely di stributed amongst various groups of

the plant kingdom and with roles in different aspects of plant development. The di scovery of oxalate oxidase activity of cereal germins enables one to visua li ze a role for a byproduct of metabolism in developmental processes of plants. The spati al and temporal distribution of gennins and related proteins highlights the ro le of cell wa ll in plant development and responses to di fferent abiotic and biotic stresses. The regulati on of germin like genes in various organi sms di splays an amazing diversity in response to similar developmental and environmenta l cues. Int roduction of oxa late ox idase gene in susceptible crop plants thus represents a promising approach for engi neeri ng of di sease res istance in plants against oxa late secreting funga l pathogen. The study of genes encoding germin(s) and genni n like protein(s) using the transgenic and the antisense approach will undoubtedl y reveal interesti ng ins ights into regu latory mechani sm of these genes and exemplify their ro le in higher plants.

Acknowledgement Financial support from the Departmen t of Bio­

technology, Go vernment of India, is gratefu ll y ack nowledged. DP vishes to thank the Council of Scienti fic and Industrial Research for the award of a SRF (NET).

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