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Journal of Cereal Science 32 (2000) 120 doi:10.1006/jcrs.2000.0315, available online http://www.idealibrary.
com on
MINI REVIEW
Structure, Biological and Technological Functions of Lipid Transfer Proteins and Indolines, the Major Lipid Binding Proteins from Cereal Kernels
J.-P. Douliez, T. Michon, K. Elmorjani and D. Marion
Institut National de la Recherche Agronomique, Laboratoire de Biochimie et Technologie des Proteines, BP 71627, 44316 Nantes cedex 03, France
Received 9 July 1999
ABSTRACT
Using specic extraction procedures, two major lipid binding protein families, lipid transfer proteins (nsLTP) and indolines have been isolated from cereal kernels. NsLTPs are basic proteins, stabilised by four intramolecular disulphide bonds and with molecular masses of 9 kDa (nsLTP1) and 7 kDa (nsLTP2). The 3D structures of nsLTP1 from barley, maize, rice and wheat seeds have been determined. Within the protein a large hydrophobic tunnel forms the lipid binding site. NsLTPs could be involved in the formation of hydrophobic cutin and suberin layers which prevent water diusion into the grain and fungal attacks. Furthermore some nsLTPs display anti-microbial activity in vitro. In brewing, barley nsLTP1, and especially its unfolded and glycosylated forms, control the formation of beer foam. Indolines are basic lipid binding proteins with a molecular mass around 13 kDa and ve disulphide bonds. Their lipid binding site is composed by an unique tryptophanrich domain. Although their 3D structure has not been determined, secondary structure and cysteine pairings suggest that indolines and nsLTP1s share similar folding characteristics. The biological role of these seed specic proteins is not known. Indolines are capable of synergistically enhancing the antifungal properties of thionins, in vitro. In their native state, indolines display good foaming properties and prevent protein foams from destabilisation by lipids. In breadmaking these proteins interfere both in dough rheology and in bread crumb texture. Puroindolines are also the main components of friabilins, proteins found at the surface of starch granules of soft wheats. Although their role in grain hardness is still obscure, their encoding genes are obviously useful for exploring the Hardness locus and manipulating grain hardness.
2000 Academic Press
Keywords: lipid transfer proteins, puroindoline, structure, cereal kernel, lipid binding, foaming, breadmaking, brewing, cutin, suberin, antimicrobial protein, hardness.
INTRODUCTION Lipids in plant seeds full dierent key functions from the storage of carbon and energy in tri-
In memory of Patrick Sodano. Corresponding author: D. Marion. Tel: (33) (0) 2 40 67 50 56; Fax: (33) (0) 2 40 67 50 25; E-mail: marion@nantes.inra.fr 07335210/00/070001+20 $35.00
glycerides to the control of exchanges with the environment in constituting the major components of biological barriers, cell membranes and external hydrophobic layers, as cutin and suberin. Lipids are also essential functional components in the processes that transform plants or plant isolates into food or feed. Notably, the role of the dierent polar and non-polar lipid fractions in breadmaking has been clearly demonstrated1.
2000 Academic Press
J.-P. Douliez et al.
The intra- and extracellular transport of these hydrophobic molecules is provided in part by specic lipid binding proteins. These macromolecules are capable of binding either monoacyl lipids such as fatty acid binding proteins (FABP) and acyl-coA (ACBP) binding proteins or both monoacyl and diacyl lipids such as lipid transfer proteins (LTP). The biological function as well as the physicochemical and structural properties of these proteins have been mainly investigated in the animal kingdom and in yeast, the eukaryote model for cell physiologists25. Plant LTPs which have been characterised so far do not share any common structural and functional properties with the corresponding animal and yeast proteins. The biological role of plant LTPs is still unknown although interesting likely hypotheses can be proposed based on our growing knowledge on their 3D structure as well as on the regulation of their genes6. Other proteins are able to interact only with natural and articial lipid aggregates such as those to be found in biomembranes, liposomes or micelles. These soluble proteins, which are also termed membrane active proteins or membranotoxins7, are widespread in the animal kingdom where they are part of the defence mechanism of animals against their predators or a means to capture their preys. The most studied membraneactive proteins of the plant kingdom are composed of the basic thionins which have a molecular mass of about 5kDa and a compact folding stabilised by three to four disulphide bonds. These proteins display strong anti-microbial properties and, in the absence of an immune system, play a key role in the defence mechanism of plants against their pathogens8,9. Recently, another protein family which has similar lipid binding properties to thionins has been discovered. These proteins have been termed indolines based on the presence of an unique tryptophan-rich domain in their amino acid sequences. They have been isolated initially from wheat seeds10 and genes encoding homologous proteins have been found in barley, rye and oats11,12. Almost nothing is known about their biological function and, in contrast with plant LTPs, studies of the functions that these proteins full in vivo are still in their infancy. Lipidprotein interactions have been suspected to play an essential role in the functionality of lipids in cereal processing. Although the rst studies were performed more than 50 years ago, it is only in the last past ve years that the major role of lipid
binding proteins in cereal technology has been highlighted. Today most of the data concern two major lipid binding proteins of cereal seeds, LTPs and indolines, although it is obvious that many other cereal proteins are able to bind and interact with lipids. In this review, we report on the recent progress that has been made on the structure and functional properties of LTPs and indolines from both physiological and technological view points with an emphasis on the wheat and barley proteins.
EXTRACTION OF PLANT LIPID BINDING PROTEINS The specic and quantitative extraction and/or isolation of lipid binding proteins is a real challenge for the biochemist. Based on the classical fractionation of wheat proteins in various aqueous and organic solvents, it is possible to isolate lipidrich protein fractions. From such studies, it was previously and too rapidly concluded that lipids interact either with gliadins and/or glutenins, i.e. the storage proteins of wheat seeds13 or with dierent soluble proteins14. However, presence of lipids in protein fractions does not necessarily mean that complexes between lipids and proteins can exist. Their presence in these fractions could be due to their own solubility and extractability in aqueous and organic solvents as well as to the structural changes of proteins in these solvents. For example, in acidic and alcoholic media, most proteins are in the molten globule state, an intermediary fold between that displayed by native and unfolded protein. The molten globule has a higher surface hydrophobicity than the native protein15 and is capable of interacting with lipids16,17. This phenomenon could explain the important changes of lipid content and composition aecting gliadin and glutenin fractions according to whether acid or alcohol is used13. Another mechanism that is generally encountered is a simple adsorption of proteins to the hydrophobic surface of oil droplets. This emulsication process is generally due to a surface denaturing of proteins which expose their hydrophobic side chains and secondary structures at the oilwater interface18. This process is probably responsible for the isolation of ligoline and S-proteins, complex mixtures of soluble proteins, from wheat dough14,19. In our approach, we dene lipid binding proteins as all proteins that in their native fold, are capable of spontaneously binding lipids. In this
Lipid transfer proteins and indolines
regard, two strategies can be used to isolate such proteins. One is based on lipid binding assays using either radio-labelled or uorescent lipids. For example, a lipid transfer assay was used to specically isolate lipid transfer proteins in wheat our extracts20. This assay consists in following the transfer of uorescent phospholipids between two membrane compartments, i.e. the donor and acceptor membranes. In this uorescence transfer assay the donor membranes are composed of pyrenylphospholipids while the acceptor membranes are composed of natural or synthetic phospholipids forming bilayer liposomes. The donor membranes are non-uorescent due to the quenching of the pyrene uorescence of closely packed phospholipid probes. On lipid transfer the phospholipid probe is gradually diluted in the acceptor liposomes and uorescence increases21. Another lipid binding assay is based on the transfer of radio-labelled phospholipids between liposomes and mitochondria, two membrane compartments easily separable by centrifugation22. The other strategy is based on the structural features of proteins which can insert into lipid aggregates. These proteins display properties closely related to those of transmembrane proteins. Non-ionic or zwitterionic detergents are frequently used to isolate such proteins23. These detergents partition into membrane bilayers and lead to the formation of mixed micelles composed of lipids, proteins and detergent molecules. In a large excess of detergent only detergentlipid and detergent protein micelles are found in the extract24. However, in a rst attempt, it is not possible to distinguish transmembrane proteins from those which are only adsorbed on the surface of membranes. The problem is especially acute when extraction is performed not on isolated membranes but on whole cells, organelles and tissues where mixed proteinlipiddetergent micelles co-exist with the soluble non-membrane proteins. Therefore, in 1981, Bordier25 developed a phase partitioning procedure that has allowed separation of detergent micelles from the bulk aqueous extract. This procedure is based on a fundamental property of most detergents to aggregate in larger micelles above a temperature called cloud point. For Triton X-114 the cloud point is about 22C and is compatible with the stability of most proteins. Practically, when the detergent extract is heated above 22C, two phases are formed after low speed centrifugation, an upper detergent-poor phase and a lower detergent-rich phase. Soluble proteins as
well as extrinsic membrane proteins are generally recovered in the upper phase while transmembrane proteins are found in the lower detergent-rich phase. Surprisingly, dierent nonmembrane proteins were recovered from TX-114 phase partitioning of wheat our proteins, such as purothionins, some amylase inhibitors and especially new proteins, puroindolines26,27. Interestingly these proteins were all able to bind lipids.
PRIMARY STRUCTURE Lipid transfer proteins are ubiquitous in the plant kingdom and today numerous sequences of these proteins are known. Those proteins which can enhance intermembrane transfer without lipid specicity are termed non-specic lipid transfer proteins (nsLTP). They form a multigenic family and more than 50 amino acid sequences of plant nsLTPs are registered in the data banks. Until now, two main families with dierent molecular masses have been isolated. One is composed by proteins of molecular mass of about 9 kDa and the other, by proteins of molecular mass of 7 kDa, referred as nsLTP1 and nsLTP2, respectively. We have chosen this numbering nomenclature dened initially for the corresponding barley proteins28,29 because it refers to known distinct families for which a lipid transfer activity has been clearly demonstrated30. In other studies the numbers, e.g. nsLTP1, nsLTP2, etc., refer to dierent isoforms of the 9 kDa type31. In contrast with nsLTP1, only few nsLTP2 sequences are known today (Fig. 1). Among the known sequences of nsLTPs, all are characterised by a conserved pattern of cysteine residues. All of these cysteine residues are involved in intramolecular disulphide bonds whose connections have been strictly conserved among nsLTP1s (see below). Regarding the aromatic amino acids, no tryptophan residues are found in the sequence of nsLTP1 and phenylalanine is rare. Two well conserved tyrosine residues are located towards the N- and the C-termini of the polypeptide backbone, respectively (Fig. 1). However, the tyrosine located near the N-terminus is replaced by a phenylalanine residue in castorbean nsLTP1s32 suggesting that the presence of a phenyl ring could be essential for the biological activity of these proteins. The tyrosine of the C-terminus region is replaced by phenylalanine in some nsLPT1s (Fig. 1) and by isoleucine and valine in the case of nsLTP1s from castor bean seedlings32.
4
Source T. aestivum T. aestivum H. vulgare H. vulgare H. vulgare Z. mays O. sativa O. sativa O. sativa O. sativa S. biocolor S. biocolor

Amino acid sequence (mature protein) 1 10 20 30 40 I D C GH V D S L V R P C L S Y V Q G G P G P S G Q C C D G V K N L HNQ A R S Q S D R Q A N C G Q V V S Y L A P C I S Y AMG R V S V P G G G C C S G V R G L N A A A A T P A D R K L N C G Q V D S KMK P C L T Y V Q G G P G P S G E C C N G V R D L H N Q A Q S S G D R Q A I S C GQ V S S A L S P C I S Y A R GNG A K P P A A C C S G V K R L A G A A Q S T A DKQ A I S C GQ V S S A L S P C I S Y A R GNG A K P P V A C C S G V K R L A G A A Q S T A DKQ A I S C G Q V A S A I A P C I S Y A R G QG S G P S A G C C S G V R S L NN A A R T T A D R R A I T CGQVNS AVG P C L T Y ARG GAGP S A A C C S GVR S L F A AA S T T ADRR A I S CGQVNS AV S P C L S Y ARG G S GP S A A C C S GVR S LNS AA S T T ADRR I S CGQVNS AV S P C L S Y ARG L R P S A A C C S GVR S LNS AA S T T ADRR V S C G D V T S S I A P C L S Y VMG R E S S P S S S C C S G V R T L N G K A S S S A D R R A I S CGQV S S A I A L C L S Y ARG G F A P S AGC C S GVR S LNS AA R T T ADRR AV T CGQV S S A I G P C L S Y ARG G S GP S AGC C S GVR S LNS AA R T T ADRR h * * *h *h * * * *h h h * * * 50 60 70 80 90 S A C N C L K G I A R G I HN L N E DN A R S I P P K C G V N L P Y T I S L N I D C S R V T T C T C L K Q Q A S GMG G I K P N L V A G I P G K C G V N I P Y A I S L N I D C S R V T V C N C L K G I A R G I HN L N L NN A A S I P S K C N V N V P Y T I S P D I D C S R I Y A A C K C L K S A A G G L N A G K A A G I P S MC G V S V P Y A I S A S V D C S K I R A A C R C L K S L A T S I K G I N MG K V S G V P G K C G V S V P Y P I S M S T D C N K V H A A CNC L KNA A A G V S G L NAGNA A S I P S K C G V S I P Y T I S T S T D C S R VN T A CNC L KNA A R G I KG L NAGNA A S I P S K C G V S V P Y T I S A S I D C S R VN T A CNC L KNV A G S I S G L NAGNA A S I P S K C G V T I P Y T I S P S I D C S S VN T A CNC L KNV A G S I S G L NAGNA A S I P S K C G V S I P Y T I S P S I D C S R VN T A C S C L K NM A S S F R N L N MG N A A S I P S K C G V S V A F P I S T S V D C S K I N A A CNC L KNA A R G I S G L NAGNA A S I P S K C G V S V P Y T I S T S T D C S R V S A A CNC L KNA A RG I R G L NVGKA A S I P S KCG V S I P Y T I S T S T D C S R V S h h* * * h * h h * h h* * h 1 10 20 30 40 A C Q A S Q L A V C A S A I L S GAK P S G E C C GN L R A QQGC F C Q Y A K A C E P A Q L A V C A S A I L GG T K P S G E C C GN L R A QQGC L C Q Y V K A P C E V G Q L T V CM A I T T G A K P S G A C C A N L G A Q Q G C F C Q Y A K V T CN P T E L S S C V P A I TGG S K P S S T C C S K L KVQE P C L CNY I K S F C RM P K D G L K S C L A S V S G D N P V D P T S D C C L A L A K A D L Q C F C R Y K 50 60 70 D P T Y GQ Y I R S PHA RD T L T S CG L A V P HC D P N Y GH Y V S S P H A R D T L N L C G I P V P HC D P A L GR Y I T S PHARDT L L S CG L A V P R C DN P S L KQ Y V N S P G A KK V L S N C G V T Y P HC S G L L S I Y G V D P N K CME L P V K C K V V D S F H C Organ
seed (aleurone) seed (embryo) seed (aleurone) leaves leaves seedling seed unknown unknown unknown unknown unknown
Accession/ref.
NLTA WHEAT NLTB WHEAT NLT1_HORVU NL41_HORVU NLT3_HORVU NLTP_MAIZE NLT1_ORYSA NLT2_ORYSA NLT3_ORYSA NLT4_ORYSA NLT1_SORVU NLT2_SORVU
nsLTP1 (9 kDa type)
T. aestivum T. aestivum H. vulgare H. vulgare H. vulgare Z. mays O. sativa O. sativa O. sativa O. sativa S. biocolor S. biocolor
seed (aleurone) seed (embryo) seed (aleurone) leaves leaves seedling seed unknown unknown unknown unknown unknown
NLTA WHEAT NLTB WHEAT NLT1_HORVU NL41_HORVU NLT3_HORVU NLTP_MAIZE NLT1_ORYSA NLT2_ORYSA NLT3_ORYSA NLT4_ORYSA NLT1_SORVU NLT2_SORVU
T. durum H. vulgare
nsLTP2 (7 kDa type)
seed (aleurone) seed (embryo) nucellus (ovule) root root seed seed (aleurone) nucellus (ovule) root root
WHEAT29 NLT2_HORVU BARLEY93 NLTP_VIGUN BEAN77 WHEAT29 NLT2_HORVU BARLEY93 NLTP_VIGUN BEAN77
H. vulgare V. unguiculata P. vulgaris T. durum H. vulgare H. vulgare V. unguiculata P. vulgaris
Figure 1 Alignment of the amino acid sequences of mature nsLTP1 and nsLTP2. For nsLTP1 only cereal proteins are aligned. (h) Hydrophobic and () other amino acid residues which are highly conserved among all plant nsLTP1 sequences available in the SWISS-PROT data bank.
This suggests that the presence of a hydrophobic residue is important in that position. Most of the hydrophobic residues involved in the formation of the hydrophobic cavity (see below) are relatively well conserved. Proline or glycine residues involved in loops and turns are also well conserved (Fig.1). Finally, in the search of a biological function it is important to locate conserved hydrophilic and/or charged residues which could play a role in a catalytic or binding site. Comparing all plant nsLTP1 sequences, these highly conserved residues are all located in two consensus pentapeptides Thr/Ser-X1-X2-Asp-Arg/Lys and a Pro-Tyr-XIle-Ser (residues 4347 and 8185 on a consensus sequence basis, respectively) (Fig. 1). Although few nsLTP2 sequences are known, it is already obvious that these conserved motifs are absent in nsLTP2
(Fig. 1). Furthermore, amino acid sequence alignment (not shown) reveals that these consensus motifs could correspond to deletion/insertion events between nsLTP1 and nsLTP2. Puroindolines are also basic and cysteine-rich proteins with molecular masses around 13 kDa. Seed puroindolines are composed of two main isoforms, puroindoline-a (PIN-a) and puroindoline-b (PIN-b). The name puroindoline was given in regard to the unique tryptophan-rich domain (Trp-Arg-Trp-Trp-Lys-Trp-Trp-Lys) of PIN-a, the rst protein which was sequenced (indoline, for the indole ring of tryptophan and puros, a greek word for wheat)10. In fact, this domain is truncated in PIN-b (Trp-Pro-Thr-Trp-Trp-Lys) the second isoform isolated26 whose amino acid sequence was deduced from cDNA sequencing 33

40 30 20 MK A L F L I G L L A L V A S T A F A Q Y S E MK A L F L I G L L A L V A S T A F A Q Y S E MK A L F L MG L L A L V A S A A F A Q Y G E MK A L F L I G L L A L V A S T A F A Q Y S E MK T L F L L A L L A L V A S T T F A Q Y S E MK T L F L L A L L A L V A S T T F A Q Y S E MK T L F L L A L L A L V A S T T F A Q Y S E MK T L F L L A L L A L V A S T T F A Q Y S E MK I F F F L A L L A L V V S A T F A Q Y V E MK A L F L L A F L A L A A S A A F A Q Q Y A D MK T F F L L A F L A L V V S T A I A Q Y A E V P
T. aestivum T. monococcum H. vulgare A. sativa T. aestivum T. monococcum H. vulgare A. sativa Trp1 (A. sativa) Trp2 (A. sativa) GSP1a (T. aestivum)
10 1 1 10 V VG S Y D V AGGGGAQQC P V E T K L V VG S Y D V AGGGGAQQC P L E T K L V VG S Y EGGAGGGGAQQC P L G T K L V VG S Y D V AGGGGAQQC P V E T K L I G GW Y N E V G G G G G S Q Q C P Q E R P K L V G GW Y N E V G A G G G S Q Q C P L E R P K L I G GW Y N E V G A G G G S Q Q C P Q E R P K L I G GW Y N E V G G G G G S Q Q C P Q E R P K L S D G S Y E E V E G P H D R C Q Q H QMK L T G V G G WD G C M P E K A R L P A A Q A P T A D G F G EWV A I A P S A S G S E N C E E E Q P K V 80 I QG S I I QG S I I QG S I I QG S I I RRV I I R GM I I R GM I I R GM I I WR S I I WR A V I WT S I
indoline-b indoline-a indoline-b indoline-a indoline-b indoline-a
N-terminal peptide signal peptide 20 30 40 50 60 70 T. aestivum N S C R N Y L L D R C S T M K D F P V T W RWW K WW K G G C Q E L L G E C C S R L G Q M P P Q C R C N I N S C R N Y L L D R C S T M K D F P V T W RWW K WW K G G C L E L L G E C C S Q L G Q M P P Q C R C N I T. monococcum D S C R N Y L L D R C T T M K D F P V T W T WW K WW K G G C E E L L H D C C S Q L S Q M P P Q C R C N I H. vulgare N S C R N Y L L D R C S T M K D F P V T W RWW K WW K G G C Q E L L G V C C S R L G Q M P P Q C R C N I A. sativa S S C K D Y V M E R C F T M K D F P V T W P T K WW K G G C E H E V R E K C C K Q L S Q I A P Q C R C D S T. aestivum S S C K D Y V M G W C F T M K D F P F T W P T K WW K G G C E H E V R E N C C K Q L S Q I A P Q C R C D S T. monococcum S S C K D Y V M E R C S T M K D F P V T W P T K WW K G G C E H E V R E K C C Q Q L S Q I A P Q C R C D S H. vulgare S S C M D Y V M E R C F T M K D F P V T W P T R WW K G G C E H E V R E K C C N Q L S Q I A P Q C R C D S A. sativa D S C R E Y V A D G C T T M R D F P I T W P W K WW K G G C E E V R N E C C Q L L G Q M P W E C R C D A Trp1 (A. sativa) N S C K D Y V V E R C L T L K D I P I T W P W K WW K G G C E S E V R S Q C C M E L N Q I A P H C R C K A Trp2 (A. sativa) GSP1a (T. aestivum) D S C S D Y V M D R C V M K D M P L T W F F P R T W G K R S C E E V R N Q C C K Q L R Q T T S R C R C K A trp-rich domain 90 100 110 120 T. aestivum Q G D L G G I F G F Q R D R A S K V I Q E A K N L P P R C N Q G P P C N I P G T I G Y YW Q G D L S G I F G F Q R D R A S K V I Q E A K N L P P R C N Q G P P C N I P G T I G Y YW T. monococcum Q G R L G G F F G F Q R D R T V K V I Q A A K N L P P R C N Q G P A C N I P S T I G Y YW H. vulgare Q G D L G G I F G F Q R D R A S K V I Q E A K N L P P R C N Q G P P C N I P G T I G Y YW A. sativa Q G R L G G F L G I WR G E V F K Q L Q R A Q S L P S K C N Q G A D C K F P S G Y YW T. aestivum Q G K L G G F F G I W R G D V F K Q I Q R A Q S L P S K C NMG A D C K F P S G Y Y W T. monococcum Q G K L G G F F G I W R D E V F K Q I Q R A Q S L P S K C NMG A D C K F P S G Y Y W H. vulgare Q S K F G G F F G I W R G D V F K Q T Q R A Q S L P S K C NMG A D C K F P S G Y Y W A. sativa Q H E L G G F F G T Q Q G L I G K R L K I A K S L P T Q C NMG P E C N I P V T F G Y Y W Trp1 (A. sativa) Q G E L G G F L G F Q Q S E I MK Q V H V A Q S L P S R C NMG P N C N F P T N L G Y Y Trp2 (A. sativa) GSP1a (T. aestivum) Q G D L S G F K G L Q Q G L K A R T V Q T A K S L P T Q C N I D P K F C N I P I T S G Y Y L C-terminal peptide
Figure 2 Alignment of the amino acid sequences of cereal indolines. Indoline-a and indoline-b sequences from dierent cereals10,12,33. Tryptophanins (Trp1, Trp2)11, grain softness protein (GSP1)35. Arrows indicate the cleavage sites of signal, extra N- and C-terminal peptides of the preproproteins obtained from the comparison of protein and cDNA sequences of PIN-a10,33.
(Fig. 2). Weak but signicant sequence homologies can be found between puroindolines and nsLTP1, except in the region containing the tryptophanrich domain34. Interestingly this domain also contains the two extra cysteine residues compared to nsLTPs. These results are a rst argument to suggest that puroindolines and nsLTP1s are structurally related. Rahman et al.35 have isolated wheat cDNAs that encode grain softness proteins (GSP) which are obviously, puroindoline-like proteins (Fig. 2) but, as yet, these proteins have not been isolated from wheat kernel. Genes encoding homologous proteins have been found in oat, barley, rye and diploid Triticum species11,12 (Fig. 2). The overall sequence identity between cereal indolines is above 85% for indoline-a and above 90% for indoline-b. Within a cereal species the identities between the a and b isoforms is about 60%. These sequence identities do not take into account wheat GSP1s35 and oat tryptophanins12. The amino acid
sequences of GSP1 and puroindolines have only 40% identity and especially many residues are changed in the tryptophan-rich domain of GSP1 (Fig. 2). In the case of oat, two types of indolines have been highlighted from cDNA cloning11 and PCR analysis of genomic DNA12, referred as avenoindolines and tryptophanins, respectively. Tryptophanins and avenoindolines display about 55% identity, values close to those existing between puroindoline-a and puroindoline-b. In contrast with GSP1, the sequence of the tryptophan-rich domain of tryptophanins and avenoindolines display high homologies (Fig. 2). In order to avoid confusion, we propose the generic name indolines for the proteins in which the tryptophan-rich domain is well conserved and GSP to designate all proteins which are closely related to the grain softness trait. GSPs include indolines and proteins with divergent sequences as GSP1 (see below). Therefore, tryptophanins should be named av-
enoindoline-c and avenoline-d (Fig. 2). Indolines are not found in maize, rice and sorghum11,12 and sequencing of the Arabidopsis genome has not yet revealed the presence of homologous proteins. Thus, indolines are restricted to the Triticeae and Aveneae tribes contrary to nsLTPs which are ubiquitous in the plant kingdom.
THE NS-LTP FOLD The algorithms that are generally used to determine the secondary structures of proteins from their amino acid sequences have predicted that nsLTP1s are all- sheets proteins36. These predictions were interesting since the all- sheet folding was also displayed by proteins of the lipocalin family4,37. Finally, FTIR, CD and NMR spectroscopies have revealed that nsLTP1 are proteins with a folding involving only -helices and turns20,38 as are ACBP39. Resolution of the three dimensional structure was rst determined from NMR and X-ray crystallography data obtained for the wheat aleurone nsLTP140 and the major maize seed isoform41, respectively. This fold is characterised by a fourhelix bundle surrounded in part by a C-terminus formed by turns that displays the shape of a saxophone (Fig. 3). The folding is stabilised by four disulphide bonds as helix H3, that contains the Cys-X-Cys motif, is linked to the N-terminal end of helix H1 and the C-terminus. On the other side helix H2, which possesses the two consecutive cysteine residues, is connected to helix H1 and Nterminal end of the C-terminus (Fig. 4). The most interesting feature of this fold is the presence of a large internal cavity. The surface of this cavity is covered with the side chain of hydrophobic residues provided by the hydrophobic faces of the amphipathic helices and by the C-terminal region. This oers a potential binding site for hydrophobic or amphiphilic ligands such as lipids. The size of this cavity can vary from one isoform to another when comparing the structures of dierent nsLTP1s4144. On rst analysis, this suggests that the structure of the natural biological ligands of nsLTP1 could be dierent between the plant species. In fact, rather than a cavity, there is a tunnel following the long axis of the protein. This tunnel is partly closed on one side by the highly conserved tyrosine which is located in the C-terminal region. Loops 1 and 3, which are composed of dierent hydrophilic and hydrophobic amino acids, partially close the tunnel on the other side. No water
molecules have been observed within the tunnel, either by X-ray crystallography or by NMR41,45. However, Raman spectroscopy of wheat nsLTP1 suggests that the tyrosine residues which are within the hydrophobic tunnel have frequencies typical of tyrosines involved in hydrogen bonds with water molecules20. Since the large tunnel cannot be empty, the possibility that numerous mobile water molecules are embedded or cross the hydrophobic tunnel has to be considered and could play a major role in lipid binding. From the crystal and NMR structures of maize nsLTP1, another small cavity has been detected at the periphery of protein structure where an embedded water molecule is probably hydrogen bonded. Although it has been not possible to determine the atoms involved in these hydrogen bonds, the candidates are Cys50, Lys54, Thr82, Ser84 and Thr8745. Among these residues, it is noteworthy that Cys50 and Ser84 are strictly conserved in plant nsLTP1. Lys54 is highly conserved but can be replaced by valine as in rapeseed nsLTP146. This trapped water molecule may play a role in packing the C-terminus to helix H3 and in preventing the collapse of the C-terminus in the hydrophobic tunnel. More intriguing is Ace-AMP1, an antimicrobial protein isolated from onion seeds47 with a fold almost superimposable on that of nsLTP148. However, even though it displays exactly the same cysteine linkages as nsLTP1, the rest of the sequence diverges markedly. For example, it possesses 19 arginines (instead of three to ve in nsLTP1), two tryptophans and a phenylalanine (absent and rare amino acid side chains in nsLTP1s). Furthermore, Ace-AMP1 is not capable of transferring lipids, probably because the tunnel is lled up with the tryptophan and phenylalanine side chains. This nsLTP1-like protein could full a quite dierent function within the plant (see below). Concerning puroindolines, the three dimensional structure has not yet been determined. However, it is known from circular dichroism and FTIR data that they have a high helix content34. Moreover, the cysteine residues display similar pairing in puroindoline and in nsLTP1s. The two extra cysteines that enclosed the tryptophan-rich domain form a disulphide bond while the eight other cysteines have nsLTP-like connections (Fig. 4). These similar arrangements of disulphide bonds support the suggestion that nsLTPs and puroindolines display similar folds. Other plant proteins that display similar cysteine motifs have an LTP
Figure 3 Structure of the wheat nsLTP140 showing the helix bundle (a) and disulphide bonds (b). Structures of barley61 (c) and maize41 (d) nsLTP1s complexed with palmitic acid. Structure of wheat nsLTP1 complexed with two lysopalmitoylphosphatidylcholine molecules55 (e) showing the highly conserved residues Arg44/Asp43/Ser40 (f ).
H1 nsLTP C C CC
H2
H3 C X C
H4 C C
H1 HPS C C
H2 C C
H3 CXC
H4 C C
H1 PIN C C C TrpRD C
H2 C C
H3 C X C
H4 C C
Figure 4
Cysteine pairing and -helix positioning in nsLTP1, HPS (hydrophobic protein of soybean) and puroindolines.
fold such as the hydrophobic protein of soybean, HPS,49 and the 2S seed storage proteins50. These proteins do not possess an hydrophobic tunnel. It is noteworthy that in these proteins, as in puroindolines, a mismatch of cysteine pairing is found for the Cys-X-Cys motif (Fig. 4). This motif is found in the corresponding H3 helix of nsLTP1 and a simple rotation of this helix may lead to a cystine mismatch. Such a rotation could be related to the absence or presence of an hydrophobic cavity in this structural family of proteins. LIPID BINDING Studies on the lipid binding properties of nsLTPs and indolines can provide useful informations to understand their biological and technological functions. It is also a way to explore the structural characteristics of these proteins. The ability of nsLTPs to enhance, in vitro, the inter-membrane lipid transfer is not apparently related to their biological function but can have consequences for cereal processing and biotechnological applications (see below). The way in which a LTP transfers lipids remains obscure. In a rst attempt, two important interactions have to be considered: (a) adsorption of the protein to the membrane interface and (b) the binding of lipids by the protein. The strength of these interactions has to be compatible with the reverse process needed for intermembrane transfer. The adsorption process has been studied using monolayer techniques.
Above a surface pressure limit of about 20 mN/ m, wheat nsLTP1 is unable to penetrate within the hydrophobic core of a monomolecular phospholipid lm. Below 20 mN/m a lipidprotein monolayer is formed at an airwater interface and a lipid molecule is bound by nsLTP1. An increase in the surface pressure initially included a transfer of the proteinlipid complex at the polar interface of the phospholipid monolayer and then an expulsion of the complex in the aqueous sub-phase51. This explains why, in the case of lipid bilayers, wheat nsLTP1 displays higher transfer activity for phosphatidylglycerol (PG) than for phosphatidylcholine (PC)52. The electrostatic dipole of the phosphoryl-choline group induces a close and strong packing of PC in bilayer vesicles. On the contrary, electrostatic repulsion between the anionic phosphoryl-glycerol led to a lower packing of lipid molecules in PG than in PC bilayer vesicles. Furthermore, the negative PG bilayer enhances adsorption of the basic nsLTP1 to the bilayer interface, a process which also disturbs the lipid packing. Therefore, for plant nsLTP1 as for other specic and non specic lipid transfer proteins of animal sources53, the transfer is mainly controlled by lipid packing. In this regard it is interesting to note that the maize nsLTP1, which displays higher transfer activity than the wheat nsLTP1, is also able to penetrate phospholipid monolayers at a higher surface pressure than the wheat nsLTP154. The binding properties of nsLTP1s have been studied mainly by structure determination of lipid
protein complexes. These studies have highlighted that the tunnel can adapt its volume for binding one or two monoacylated lipids41,42,55 or a diacylated lipid56. Other hydrophobic molecules such as organic solvents (cyclohexane, benzene, etc.)57 and prostaglandine B2 (PGB2)58 can be bound in the tunnel. The signicant increase of the volume of the tunnel on lipid binding shows that it has a high plasticity and that it is impossible to predict or interpret the binding properties of plant nsLTP1s on the basis of the volume of the tunnel of the free protein. For polar lipids, the complex is stabilised by hydrogen bond between the tyrosine of the C-terminal region and the lipid phosphate or carboxylate group. Moreover, these anionic groups are in close contact with the arginine residue that is highly conserved in nsLTP1s (Asp-Arg consensus motif ) (Fig. 3). This favours the interaction between the anionic lipid and the protein. This Arg residue could be an alternative for binding of anionic lipids when the C-terminal tyrosine is absent as in castor been nsLTP132. Interestingly, in this Asp-Arg motif, the lateral chain of the aspartate is oriented at the opposite side, far from the lipid ester bond. This aspartate residue could be responsible, in conjunction with His35, for the low but signicant esterase activity of wheat aleurone nsLTP159. An interesting question deals with the orientation of these Arg and Asp residues when the polarity of the aliphatic chain is changed, e.g. a hydroxyl instead of a carboxyl/ phosphate. One can suggest that this motif could act as a switch for binding of lipids of dierent polarity. As discussed further, this is especially important in the case of the putative role of nsLTP1s in the transport of cutin monomers (fatty acids, mono, di and tri-hydroxy-fatty acids). With regard to this function, one can wonder how these lateral polar hydroxyl groups are accommodated within the hydrophobic tunnel. A rst response is provided by the structure of the nsLTP1-PGB2 complex58 where Ile81 undergoes a large conformational change to prevent unfavorable contacts with the hydroxyl group of the aliphatic chain of PGB2. In addition, this hydroxyl group, located in the centre of the aliphatic chain, becomes accessible to the solvent. So, this suggests that dierent outlets can be managed within the tunnel to allow binding of fatty acids with lateral polar hydroxyl groups. In the case where two molecules of lyso-phosphatidylcholine are bound by wheat nsLTP155, the second lipid adopts a dierent orientation. Its polar head protrudes outside the
protein between helix H1 and H3 (Fig. 3). A similar orientation was observed in the case of barley complexed with a palmitoyl-coenzyme A or a palmitic acid molecule60,61. This is rather surprising since every structure of nsLTP1 complexed with only one lipid shows an orientation with the polar head protruding between loop 2 and the C-terminal region, that is, at the opposite side (Fig. 3). In addition, the barley nsLTP1 possesses both the Asp-Arg motif and the tyrosine residue in the C-terminal region so that the dierence in orientation of the lipid suggests another mechanism of interaction. Indeed, other amino acids in the barley nsLTP1 could stabilise the lipid in that position. Finally, it is also noteworthy that the well conserved N-terminal tyrosine residue never seems involved in the stability of lipid protein complexes studied until now. These structures of nsLTP1-lipid complexes give information on how the lipid can enter within the protein. As mentioned above, nsLTP1s can bind vesicles or micelles so that the nature of the rst interaction between the protein and the lipid is polar or electrostatic. It has been proposed that the C-terminal region could play a role in the binding process because few amino acids appeared to be mobile in the crystal structure41. We propose that the mechanism of binding involves an opening of the C-terminal region exposing the hydrophobic cavity. The lipid could then be sucked up within the protein, its polar head crossing the nsLTP1. This model has the advantage of providing a uniform mechanism independent of the lipid concerned. The lipid could exit the protein by a reverse process or by continuing to cross the protein to be expulsed in the solvent. In a rst attempt, the presence of both water molecules and hydrophobic residues in the cavity should help the protein to transfer and bind molecules that have both hydrophilic and hydrophobic groups. It is therefore obvious that the challenge in the future should be the study of the rearrangements undergone on lipid binding by both water molecules and hydrophobic side chains within the tunnel. Even though these structural studies provide very valuable information, they do not allow us to quantify the stability of the complex. Experiments were performed by competition of dierent lipids with anthroyloxy or pyrene labelled uorescent lipid analogues62,63. From these binding experiments, it has been suggested that nsLTP1s are capable of binding two monoacylated lipids in agreement with structural data obtained by X-
10
ray crystallography. However, in these binding competitions, the anity of lipids for the protein cannot be determined. The anity of the protein for the lipid can be characterised by the dissociation constant, Kd, in the equilibrium: lipid+nsLPT1lipid in nsLTP1. This can be measured by following the increase of intrinsic tyrosine uorescence on lipid binding and the resulting titration curve can be tted with models that return both the dissociation constant (Kd) and the number of lipid molecules bound by the protein42,56,64. Only the tyrosine located in the Cterminal region participates in this enhancement of uorescence intensity65. The number of lipid molecules bound as determined by the tting procedure is between one and two conrming that two lipids can be loaded within nsLTP1. The Kd is always in the micromolar range for diacylphospholipids56 and monoacyl-lipids with a chain length above 12 carbon atoms59,64. In contrast with nsLTP1s, the interaction of puroindolines needs the presence of an interface. For example, in the case of lysophosphatidylcholine, phospholipids forming micelles in solution, interaction occur only above the critical micellar concentration66. About four to ve binding sites are found on puroindolines in accordance with their capacity to insert more or less deeply into lipid aggregates such as micelles or vesicle bilayers6668. Furthermore, this high number of binding sites is not compatible with the presence of a cavity within the protein. As observed for nsLTP1, these basic proteins interact more with anionic phospholipids than with neutral polar lipids such as phosphatidylcholine or galactosylglycerides68,69. Furthermore, an important dierence between the interactions of PIN-a and PIN-b is observed, which is probably due to the loss of tryptophan residues in the corresponding domain66,6870. However, it is interesting to note that PIN-b interacts more strongly with anionic lipids than does PIN-a68. Since PIN-b displays a higher positive net charge than PIN-a, these results suggest that electrostatic forces contribute to the interaction of puroindolines with polar lipids. The important role of tryptophan and cationic residues has been already emphasised for other proteins and peptides which insert in lipid bilayers7173. BIOLOGICAL FUNCTIONS The biological function of both nsLTP and puroindolines is still unknown. Initially, nsLTPs were
selected from a screening procedure for proteins involved in the intracellular ow of membrane lipids74, as previously carried out with dierent animal cells75. In all plant species and organs, the major response in the transfer assay coincided with proteins displaying a molecular mass around 9 kDa by SDS-PAGE. The ubiquity of such proteins was in good agreement with their postulated role in the intracellular transport of lipids. The rst evidence that nsLTP1s were not involved in intracellular lipid exchange or transfer has arisen from the discovery that nsLTP1s are synthesised as pre-proteins containing a signal peptide of 2025 amino acids at the N-terminus of the mature protein36. This signal peptide suggested that nsLTP1s entered the secretory pathway, a result inconsistent with a role in the cytoplasm of cells. This pathway was conrmed by the discovery that nsLTP1s could be located outside cells, on the cell walls76 and secreted into the culture medium of embryogenic cells77,78. Therefore, Sterk et al.77 have suggested that nsLTP1s are involved in the transport of hydrophobic monomers (fatty acids, fatty alcohols and hydroxy-fatty acids) which compose the waxy and polymeric cutin layers of most aerial organs79 (Fig. 5). This hypothesis is in agreement with the ability of nsLTP1s to bind all sort of monoacylated lipids with similar anities64. This is also strengthened by the fact that nsLTP1 genes are mainly expressed in epidermal tissues of plants80,81 and, in some cases, nsLTP1 can be isolated from surface waxes82. Furthermore, a relationship has been observed between an increase of the thickness of the cuticle wax layer and overexpression of an nsLTP1 gene in the leaf epidermis of barley83. However, it could be argued that some nsLTPs8486 are synthesised in roots, cutin-free organs87. Though integuments of some organs, especially underground ones, do not contain cutin they are composed of another hydrophobic polymer, suberin79. Suberin is a complex polymer composed of phenolic compounds dicarboxy- and hydroxy-fatty acids (Fig. 5). In this regard, it is worth noting that root nsLTPs belong to the nsLTP2 type8486 while the genes encoding nsLTP1s are weakly or not expressed in this organ77,80,88,89. On the contrary, both nsLTP1s and nsLTP2s are present in organs, such as seeds2830, 44,86 where both cutin and suberin layers are synthesised90. In Rhizobium-induced nodules suberinlike material is observed91 while the biosynthesis of nsLTP2 is induced84. Therefore, these results could suggest that nsLTP1s are involved in the
11
Suberin monomers CH3(CH2)mCOOH CH3(CH2)mCH2OH HOCH2(CH2)nCOOH HOOC(CH2)nCOOH Phenolics (m = 1830; n = 1420)

cell wall carbohydrates
O O C CH2 CH CH O CH2 O C CH O CH C O O CH2 O C CH CH2 O CH2OH CH3O O CH CH O CH2 O C CH CH CH2 O C O O CH3 CH3
Cutin monomers C16 family CH3(CH2)14COOH HOCH2(CH2)14COOH HOCH2(CH2)xCHOH(CH2)yCOOH (y = 8,7,6, or 5 x + y = 13) C18 family CH3(CH2)7CH = CH(CH2)7COOH HOCH2(CH2)7CH = CH(CH2)7COOH HOCH2(CH2)7CHOH-CHOH (CH2)7COOH HOCH2(CH2)7CH-CH(CH2)7COOH O
O O C-O-CH2(CH2)5-CH(CH2)8-C (CH2)8 CH-OH (CH2)5 O C O O CH2 (CH2)5 O CH-O-C-(CH2)14CH3 (CH2)8 (CH2)5 C O
(CH2)8 CH-OH
CH2
(CH2-O-C-(CH2)14CH2-O O O O-(CH2)8-CH-(CH2)5CH2O-C-(CH2)8CH(CH2)5CH2-OOH O O C O OH OCH3 CH CH O O n
O O
CH CH2 CH2 O C CH CH
CH3O OH OH
Figure 5
Major components (upper) and model structures of cutin (right) and suberin (left) polymers. From Kolattukudy79.
transport of hydrophobic cutin monomers while nsLTP2s are involved in the transport of hydrophobic suberin monomers. NsLTP2s are also synthesised during the earlier stages of tracheary element dierentiation from isolated Zinnia elegans leaf mesophyll cells92 as well as in the outer layer of the nucellus of the barley ovule after pollination93. However nothing is known on the biosynthesis of polymeric and hydrophobic layers during these particular physiological stages. A role of nsLTP1 in the formation of sporopollenin, a -carotenoid, xanthophyll and fatty-acid polymer specic to the pollen wall has also been suggested94. The role of nsLTPs in the formation of protective and hydrophobic surface layers of plant organs is compatible with observations that nsLTP synthesis increases in case of abiotic stress (salt, drought, cold, wounding)9597. However, it has been shown that some nsLTP1s can be identied in the glyoxysomes and whole cotyledons of castor bean seeds98,99 and rape seedlings100 and within the aleurone grains of wheat seeds101. Since the latter ob-
servations concern organs or cells involved in the storage of lipids, it has been suggested that nsLTPs are also involved in the transport of fatty acids or their CoA derivatives on lipid mobilisation99,100. A dual function in cutin/suberin biosynthesis and in lipid mobilisation is possible in regard to the ability of nsLTPs to bind all types of fatty acids. However, other observations do not argue for a role in lipid mobilisation. For example, the biosynthesis of most nsLTPs is stimulated by abscisic acid83,95,97, a plant hormone involved in seed dormancy102, while the wheat aleurone nsLTP1 is degraded on lipid mobilisation101. This debate on the biological function of nsLTPs started again with the discovery that some nsLTPs were eective in inhibiting the growth of plant pathogenic bacteria and fungi in vitro and that microbial pathogens induced an over-expression of the genes encoding nsLTPs31, 103 . Furthermore, transformation of tobacco with genes encoding anti-microbial isoform of nsLTP confer resistance to the plant104. However, some nsLTPs, such as the wheat aleurone isoform, is
12
devoid of anti-microbial activity, although it is capable of synergistically enhancing the anti-microbial properties of wheat thionins101. The fact that nsLTP could be a part of the defence mechanism of plant against microbial pathogens is compatible with a role in the formation of external hydrophobic layers. In this respect, it has been suggested that suberisation occurs on attack by micro-organisms as well as after wounding even on tissues normally covered by cutin79. These results suggest that the anti-microbial activity of nsLTPs could be secondary to their primary biological function. However, even though the nsLTP1-like antifungal protein, Ace-AMP1, is able of interacting with lipid bilayers and of inducing permeability changes, it is unable to bind lipids within its hydrophobic core48. As a consequence, such protein is certainly not involved in the transport of cutin/suberin monomers. As mentioned above the amino acid sequence of Ace-AMP1 diverges strongly from the consensus sequence of other nsLTP1s. Furthermore, in contrast with nsLTPs, Ace-AMP1 is synthesised as a pre-proprotein with a N-terminal extension of 12 amino acids between the signal peptide and the mature protein47. Therefore, with the loss of the hydrophobic cavity, it is possible that some plant nsLTP1s have evolved to lose their primary function (formation of hydrophobic layers) and to retain and increase their secondary antimicrobial activity. Comparison of Ace-AMP1 with other anti-fungal and non anti-fungal nsLTPs suggests that the membrane permeabilising and lipid transfer properties are not related. Discovery of a true nsLTP1 together with Ace-AMP1in onion seeds would be informative on the evolutionary behaviour and functionality of this structural family of proteins. Finally, the role of nsLTPs in the formation of the hydrophobic layers that protect organs from physical and biological stress is today the most convincing hypothesis inasmuch as these hydrophobic polymers and nsLTPs are both restricted to the plant kingdom. This is also in agreement with the high amounts of these proteins in the soluble protein fractions of most plant organs. To explore this attractive hypothesis, the mechanism of polymerisation of cutin monomers, which is still unknown, has to be investigated. Especially the corresponding enzyme or enzyme complex, i.e. cutin polymerase79 has not been isolated yet. As with Ace-AMP1, puroindolines are synthesised as pre-proproteins containing an N-ter-
minal signal peptide followed by a sequence of eight to 10 amino acids33. Furthermore, an aromatic tripeptide (Tyr-Tyr-Trp) is cleaved from the C-terminus of the mature protein (Fig. 2). This post-translational processing is quite dierent from that observed for nsLTPs and could play a specic role in cell routing and function. In wheat kernel, puroindolines are recovered in the protein matrix or remain associated with membrane remnants101 which are found between the protein matrix and starch granules105107. Using specic monoclonal antibodies it has been possible to show that PINa is mainly located in the starchy endosperm101. The synthesis of PIN-a begins early during grain maturation similar to that of storage proteins, in agreement with their probable localisation in protein bodies101,108. Until now it has not been possible to localise unambiguously the b-isoform since the corresponding antibodies were not eective in immuno-cytochemistry. The results available today indicate that PIN-b is located either only in aleurone cells or in both aleurone and starchy endosperm cells101. The latter hypothesis is in agreement with recent studies using reporter genes109. Puroindolines are found both in the aleurone layer and starchy endosperm of wheat kernel but are not found in leaves and roots suggesting that they are seed specic proteins33. In aleurone cells, puroindolines are localised in the aleurone grain and apparently not in the same type of inclusions than nsLTP1. Furthermore, they are located mainly at the periphery of these inclusions. Therefore, puroindolines follow the secretory pathway and are probably stored in protein bodies, most probably associated with the membrane of these organelles. This hypothesis is in good agreement with the fact that both PIN-a and PIN-b can be extracted only with membrane breaking agents, e.g. non-ionic detergents26 or propan-1-ol110, similar to membrane proteins. Together with prostaglandine synthetase111, puroindolines could be among the rare examples of monotopic membrane proteins. However, the function of these putative membrane proteins in protein bodies is still unknown. In contrast with nsLTPs, indolines are not ubiquitous and appear to be restricted to some monocotyledones11,12 where they probably full a specic function. The sole biological activity which has been highlighted today, in vitro, for puroindolines is antifungal101. As for antimicrobial nsLTPs, the antifungal properties of puroindolines are probably related to their membrane permeabilising prop-
13
erties as recently observed on neurones112. As for the antimicrobial peptides, indolicidin113 and tritrpticin114, the role of both the tryptophan-rich domain and positive net charge of puroindolines are probably very important in their antifungal activity. Puroindolines are also capable of synergistically enhancing the antifungal properties of thionins. From a biological point of view such a synergy is more signicant than those observed between nsLTP1 and thionins101 since PIN-a and thionins are both located in the starchy endosperm and at the periphery of protein bodies101,115. However, not enough information is available on the regulation of their genes, especially in response to fungal infection to denitely conclude that puroindolines play a role in the defence mechanism of plants against their pests and pathogens. Nevertheless, it is highly probable that, as discussed for nsLTPs, this antifungal activity is a secondary function for puroindolines.
LIPID BINDING PROTEINS AND CEREAL PROCESSING NsLTPs and puroindolines are surface active proteins since they are capable of adsorbing spontaneously at airwater interfaces51,116. However, only puroindolines are able to form very stable foams. The stability of puroindoline foams is intrinsically high, for example about 10 times as high as that of egg white protein foams68. However, the most interesting feature of puroindoline foams is their high resistance to destabilisation by both neutral and polar lipids6668,117. Furthermore, a synergistic enhancement of the stability of puroindoline foams can occur in the presence of polar lipids6668. Such a synergy is unique since polar lipids and surfactants generally compete with proteins for airwater interfaces18. This phenomenon is closely related to the anity of puroindolines for lipids and, for example, this synergy is less pronounced for PIN-b which interacts less strongly with most polar lipids66,68. These strong surface-active properties are also expressed in complex system such as beer and bread117,118. In beer, it has been shown that small amounts of puroindolines can restore foam destabilised by dierent neutral and polar lipids117. In bread, puroindolines lead to crumb with a homogeneous structure composed of ne gas cells. These eects were observed by adding relatively small amount of puroindolines (0.05 to 0.2%) to
ours obtained from PIN-a-free cultivars118. The ne gas cells of bread crumb are quite similar to the ne bubbles of puroindoline foams. Preliminary experiments suggest that puroindolines act on the foaming properties of aqueous phase of dough by preventing foam destabilisation by non polar lipids and also by their capacity to synergistically increase the surface properties of wheat polar lipids119. Concerning bread volume, an increase or a decrease can be obtained according to the rheological properties of the wheat our dough. The dough viscoelasticity can interfere with gas cell growth on mixing, fermentation and baking. This interference is relatively complex since, surprisingly, puroindolines also induced some important changes in the rheological properties of wheat our dough. Thus, it has been shown that addition of puroindolines to wheat our induces an increase in dough tenacity and a decrease in dough extensibility118. These changes can be related to the concentration of puroindolines in the gluten fraction of dough119. However, this result is in contradiction with previous results showing that extraction of lipoproteins from wheat gluten does not change gluten viscoelasticity120. These contradictory results can be reconciled if we consider that puroindolines interfere on the formation of gluten occurring on mixing. A possible hypothesis is that puroindolines activate enzymes which function at waterlipid interfaces as lipases and lipoxygenases. These enzymes are known for their ability to change dough rheology121. Nevertheless, it is important to keep in mind that such reconstitution experiments do not necessarily predict what would occur with naturally present puroindolines. Although the use of isogenic lines for puroindolines would be probably necessary to go further with such studies, it is clear that these lipidbinding proteins are ecient ingredients to change the texture of baked products. Concerning the barley aleurone nsLTP1, it has been shown that this protein concentrates in beer foam. This beer foam nsLTP1 contributes to foam formation while protein Z, a barley albumin of higher molecular mass, confers foam stability122. In fact, the native barley seed nsLTP1 displays poor foaming properties and becomes a foam promoting agent only after unfolding on wort boiling123. Recently it has been shown that beer nsLTP1 is glycosylated due to Maillard reactions that occur probably on malting124. Glycosylation could hinder protein precipitation on unfolding that occurs on wort boiling. Furthermore both
14
glycosylation and unfolding should lead to increase the amphiphilic character of nsLTP1 polypeptides and contribute to a better adsorption at airwater interfaces. It has been also suggested that nsLTPs is an inhibitor of cysteine endoproteases in green malt125. However, this inhibitory activity is observed only after protein unfolding and proteolysis of barley nsLTP1 after heating. This could be a competitive inhibitory eect since nsLTP1 polypeptides are better substrates for cysteine endoproteases than hordeins126. The role of nsLTP1 in breadmaking is still unknown. During mixing and fermentation which are relatively mild technological treatments, the wheat nsLTP1s should remain in its folded state. Therefore as previously suggested1 nsLTPs could facilitate the transfer of wheat polar lipids as membrane bilayers in the bulk aqueous phase to monolayer at airwater interfaces.
PUROINDOLINES AND KERNEL HARDNESS The texture of the wheat endosperm, termed hardness or softness, is a rst discriminating characteristic for the end uses of wheat seeds. Generally hard wheat is used in breadmaking while soft wheat is used for making cookies, biscuits and pastries. Grain hardness is controlled primarily by a major locus, referred as Hardness (Ha), though softness is in fact the dominant trait. The Ha locus is located on the short arm of chromosome 5D in hexaploid wheat127. No biochemical marker was known until Greenwell and Schoeld128 reported a relationship between the presence of a 15 kDa protein observed by SDS PAGE, named friabilin, on the surface of water-washed starch and grain softness. In fact, friabilin is a complex mixture of proteins including puroindolines and GSP1 as the major components27,108,129. Puroindolines are present in most hard and soft wheat although there is a tendency for lower amounts to be present in hard wheat endosperm118. For example, PIN-a free cultivars are always hard or very hard wheat118,130. As the Ha locus, the PIN-a gene is located on the short arm of chromosome 5D while GSP-1 genes are present on the homeologous chromosome of the A, B and D genomes of hexaploid wheat (AABBDD)131133. Recent studies have shown that, although PIN-a and PIN-b genes are absent from tetraploid wheats (AABB), they are found in diploid species supposed to be the donors of A and B
genomes12. This striking result is probably due to deletion of the corresponding genes in tetraploid wheats. They have been restored by hybridisation of T. turgidum with T. tauschii. This hypothesis is in agreement with the total identity of PIN-a and PIN-b coding sequences12 and with the rapid elimination of low-copy DNA sequences during wheat speciation through allopolyploidy134. The location of PIN-a and PIN-b genes on the short arm of chromosome 5A and 5D of diploid wheats133,135 suggest that these genes are located on the homoelogous chromosome 5B of corresponding diploid Triticum species. The restriction map of the corresponding BAC clone shows that the Pina-Am1 was located between the Pinb-Am1 and the Gsp1Am1 genes (Fig. 6). The high degree of homology found between the puroindoline and GSP1 sequences in T. monococcum and T. aestivum as well as their similar chromosome location argue for a similar organisation of related genes on chromosome 5D of T. aestivum. An interesting allelic variation has been observed that leads to a glycine to serine mutation (Gly46Ser) in PIN-b. This allelic variation is always associated with hardness130,136. When the Gly46Ser mutation is not observed in a hard wheat, it has been proposed that this is always associated with the null PIN-a variants130. However, this conclusion has not been totally conrmed for an Australian wheat sample in which some hard cultivars have the soft PIN-b allele while PIN-a is present137. These contradictory results show that the role of puroindolines in wheat hardness is not so simple as it could be expected from the discovery of friabilins and identities between puroindolines, GSP1 and friabilins. They also emphasise our lack of knowledge on the physicochemical basis of grain hardness and obviously, of its measurement. It is highly probable that many interacting genes on the Ha locus are responsible for determining endosperm texture. This is illustrated, for example, by the tight linkage between the Ha locus and the Fpl1 locus that controls the free polar lipid content of wheat our138. Nonetheless, puroindolines are the only marker available for further investigation of the structure and function of the Ha locus.
CONCLUSION Besides gluten proteins, the lipid transfer proteins and puroindolines are the wheat proteins for which we have the most exciting information on struc-
15
Physical map
Pc
Pm
As
As
As
As
Pm
Pm N
Pc
vector 10 kb Pinb-A 1
m
vector
Pina-A 1
Gsp-A 1
Genetic map To centromere
1 cM Xmwg920 XNor
Figure 6 Physical and genetic maps of the Ha related gene region on the short arm of chromosome 5A of T. monoccocum. The physical map is based on the digestion with combinations of restricted enzymes NotI (N), AscI (As), PacI (Pc) and PmeI (Pm). Restriction fragments containing Gsp-Am1, Pina-Am1 and Pinb-Am1 genes are indicated by lled and hatched areas. Xmwg920 and XNor are anking loci on short arm of chromosome 5Am (from Tranquilli et al.135).
ture-function relationships. Unexpectedly, nsLTPs and puroindolines probably share structural features so that we can dene that we call the nsLTP fold. Such a fold is also more or less displayed by other non-lipid binding proteins such as HPS, amylase protease inhibitors and 2S storage proteins from oil seeds. This large structural family strengthens previous hypothesis on the evolution of some plant cystine-rich proteins including the non repetitive domains of gliadins139. In the near future, many studies will have to be done to determine the biological functions of these lipid binding proteins. In the case of the ubiquitous nsLTPs, the complete sequencing of the Arabidopsis and rice genomes as well as structural investigations on nsLTP2 should bring new essential data. For puroindolines the complete sequencing and regulation of the genes encoding these proteins is underway in dierent laboratories. These studies are required to approach the biological function of puroindolines. Nevertheless, nsLTPs and puroindolines have already great prospects both for agronomy to improve resistance to microbial pathogens and for technology to improve the end
uses of wheat and other cereal species. With puroindolines, we have the rst alternative to storage protein for manipulating wheat breadmaking quality while with nsLTP1 we have an opportunity to improve the malting and brewing quality of barley. This opens new perspectives for future plant breeding programmes. However, before such developments take place great care must be taken to ensure that there is no associated risk of allergy since it has been recently shown that the major allergens of some fruits are nsLTP1140,141.
Acknowledgements
We are indebted to the Institut National de la Recherche Agronomique, the Ministere de lEducation ` Nationale de la Recherche et de la Technologie, the Region Pays de la Loire and the European Community (FAIR programme) for supporting this research. We are grateful to our colleagues G. Branlard, W. Broekaert, D. C. Clark, C. Cohen-Addad, E. Forest, M. F. Gautier, V. Lullien, E. Pebay-Peyroula, M. Pezolet, M. Ptak, S. Rahman, P. Sodano, F. Vovelle and P. J. Wilde for fruitful contributions and stimulating discussions
16
along this work. We thank I. Altosaar, J. Dubcovsky, J.-C. Kader, E. A. Pastorello for providing unpublished data and pre-prints. We also thank M. Coupa for kindly helping to review this manuscript and C. Nicolas for preparing photographs.
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Journal of Cereal Science 32 (2000) 120 doi:10.1006/jcrs.2000.0315, available online http://www.idealibrary.

J.-P. Douliez et al.

Lipid transfer proteins and indolines

J.-P. Douliez et al.

nsLTP1 (9 kDa type)

H. vulgare V. unguiculata P. vulgaris T. durum H. vulgare H. vulgare V. unguiculata P. vulgaris

Lipid transfer proteins and indolines

indoline-b indoline-a indoline-b indoline-a indoline-b indoline-a

J.-P. Douliez et al.

Lipid transfer proteins and indolines

J.-P. Douliez et al.

Lipid transfer proteins and indolines

J.-P. Douliez et al.

Lipid transfer proteins and indolines

Suberin monomers CH3(CH2)mCOOH CH3(CH2)mCH2OH HOCH2(CH2)nCOOH HOOC(CH2)nCOOH Phenolics (m = 1830; n = 1420)

O O C-O-CH2(CH2)5-CH(CH2)8-C (CH2)8 CH-OH (CH2)5 O C O O CH2 (CH2)5 O CH-O-C-(CH2)14CH3 (CH2)8 (CH2)5 C O

(CH2-O-C-(CH2)14CH2-O O O O-(CH2)8-CH-(CH2)5CH2O-C-(CH2)8CH(CH2)5CH2-OOH O O C O OH OCH3 CH CH O O n

J.-P. Douliez et al.

Lipid transfer proteins and indolines

J.-P. Douliez et al.

Lipid transfer proteins and indolines

Genetic map To centromere

J.-P. Douliez et al.

Lipid transfer proteins and indolines

J.-P. Douliez et al.

Lipid transfer proteins and indolines

J.-P. Douliez et al.

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