Inflorescence bud proteins of Pistacia vera

Trees (1998) 12:415–419

© Springer-Verlag 1998

O R I G I NA L A RT I C L E

&roles:Avi Golan-Goldhirsh · Irena Peri · Yehudith Birk Patricia Smirnoff

Inflorescence bud proteins of Pistacia vera

&misc:Received: 17 October 1997 / Accepted: 6 March 1998

&p.1:Abstract The Pistacia vera L. (common name pistachio) is a unique dioecious and deciduous tree species, which is productive under harsh desert climates. We have identified and purified an Inflorescence Bud Protein of 32 kDa (IBP32) from male pistachio trees. There is a close correlation between its accumulation and inflorescence bud development and its disappearance and flowering. Using antibodies raised against this protein, we have identified in female trees the IBP32 and in addition a 27 kDa protein (IBP27), which appears to be specific to female inflorescence buds. The accumulation and disappearance of IBP27 follows the same pattern as that of IBP32. These proteins are glycoproteins rich in glycine and alanine and are highly hydrophilic. Based on the analytical results and immunological cross-reactivity between dehydrin antibodies and the IBPs, it is assumed that the latter are dehydrin-like and may protect inflorescence bud meristems against cold injury during dormancy. The IBPs are the major proteins of the pistachio bud, therefore they may also serve as nitrogen storage during winter for inflorescence bud growth in spring. &kwd:Key words Inflorescence bud proteins · Antibodies · Dormancy · Deciduous · Dioecious&bdy:

Introduction Flower formation in higher plants is a complex process controlled by genetic as well as environmental factors (Bernier 1988). Although it is an integrated process, three major phases can be recognized in deciduous trees: A. Golan-Goldhirsh (✉) · I. Peri Ben-Gurion University of the Negev, The Jacob Blaustein Institute for Desert Research, The Albert Katz Center for Desert Agrobiology, Desert Plant Biotechnology Laboratory, Sede Boker Campus 84990, Israel Y. Birk · P. Smirnoff The Hebrew University of Jerusalem, Institute of Biochemistry, Food Science & Nutrition, Rehovot 76100, Israel&/fn-block:

floral induction, bud dormancy and flowering (Bernier 1988; Martin 1991). Metabolic and biochemical changes have been studied extensively during the annual growth cycle of numerous woody plant species. However, little is known about the molecular processes involved in entry and exit from dormancy (Martin 1991). A new group of proteins termed bark storage proteins (BSPs) has been reported in Malus, Sambucus, Robinia, Acer, Salix, Populus, Prunus and other deciduous trees (Stepien et al. 1994). Nitrogen stored in the BSP pool is remobilized during spring shoot growth and is a significant contribution to plant nutrition (Coleman et al. 1993). In some cases homology between BSP and dehydrins has been shown (Wisniewski et al. 1996; Arora and Wisniewski 1994). The dehydrin family contains proteins which accumulate in plants under drought, cold, salinity, short day and during dormancy (Arora and Wisniewski 1994; Close et al. 1993; Sauter and van Cleve 1990; van Cleve and Apel 1993). The pistachio (Pistacia vera L.), as a deciduous and dioecuous fruit tree, offers unique developmental characteristics, i.e., flower bud dormancy in combination with chilling requirement for flowering and distinct separation of the sexes (Whitehouse and Stone 1941; Golan-Goldhirsh et al. 1991). Macroscopic and microscopic aspects related to the inflorescence development of male and female pistachio were described in the literature (Grundwag and Fahn 1969; Takeda et al. 1979; Crane and Iwakiri 1981; Golan-Goldhirsh 1995). In order to relate the phenological development of inflorescence bud to gene expression, bud development can be presented by two major bursts of growth shown schematically in Fig. 1. Differentiation of a vegetative axillary meristem into a reproductive bud occurs on male trees during April. The bud reaches its ultimate size by late June (First burst of growth, Fig. 1). This will vary slightly with cultivar, environment and degree of winter chilling. Flowering of these buds starts in the last week of March of the following year and anthesis occurs commonly during the first half of April (Second burst of growth, Fig. 1). The developmental pattern

416

Fig. 1 Approximate timing of Pistacia vera L. male and female inflorescence bud development&ig.c:/f

discarded and the supernatant was used for SDS-PAGE analysis and for further purification. Purification of the IBP32 was performed by adding cold acetone at a ratio of 1 to 4 (v/v). The formed precipitate was allowed to settle for 15 min at 4°C, separated by centrifugation as before and the residual acetone evaporated in the air stream. It was then dissolved in a volume of deionized-distilled water equivalent to that used for extraction, centrifuged at 10 000 g for 10 min and the supernatant was used for further purification by HPLC on Superdex 75-HR column or by electroelution from SDS-PAGE, as follows. The supernatant was applied to the Superdex 75-HR 10/30 column (Pharmacia), pre-equilibrated with 50 mM Na2HPO4 H2O containing 150 mM NaCl, pH 7.4. Proteins were eluted with the same buffer at a flow rate of 0.5 ml/min. Proteins were detected in the eluent by optical density at 280 nm. The IBP32 containing fractions were collected, dialyzed against deionized-distilled water and freeze-dried. Alternatively, the supernatant was separated by SDS-PAGE according to Laemmli (1970) on a 10% polyacrylamide gel. The IBP32 was cut-out from the gel and electroeluted on an ElectroEluter (Bio-Rad, model 422). The purified protein was used to prepare polyclonal antibodies according to the procedures described by Livingston (1974). Characterization of IBP32

of female buds is similar to that of male buds, except that in the female the initial inflorescence differentiation into rachis, lateral branches and sepals takes place between April and June, followed by a quiescent period of about 3 months; then pistil growth commences in early October and continues slowly until March, when individual flowers open (Takeda et al. 1979). Little is known about the proteins and enzymes which are produced in the inflorescence bud of deciduous species during dormancy and flowering. In this paper we describe major proteins IBP32 and IBP27, which accumulate in the bud of P. vera during dormancy.

Materials and methods Plant material Inflorescence buds of pistachio (Pistacia vera L.) were collected at several stages of development at the experimental orchard of Avdat Run-Off Farm in the Negev Desert of Israel. Upon harvest, buds were immediately frozen in liquid nitrogen and stored at –80°C for preparation of tissue powder. Extraction and purification of the IBP32 protein Frozen buds were ground in a mortar while still in liquid nitrogen. The powder was extracted with cold acetone in a ratio 1:4 (w/v) 3–4 times and the defatted powder was air-dried overnight. The dry material was powdered in a coffee blender and stored desiccated at 4°C (tissue powder). Preliminary experiments have shown that strong denaturing conditions, including SDS and reducing agents in the extraction buffer, are necessary to obtain high yield of bud proteins. The following protocol was chosen for the extraction of the IBP32 protein (Golan-Goldhirsh et al. 1990): the tissue powder was suspended in a ratio of 1:4 (w/v) in 62.5 mM TRISHCl buffer pH 6.8, containing 2% SDS, 10% glycerol, 2.5 mM Na2S2O5, 5 mM ascorbic acid, 2 mM PMSF, 21 µM leupeptine, 2 mM EDTA and 5% β-mercaptoethanol. The suspension was stirred for 15 min at room temperature, boiled for 5 min, cooled and centrifuged at 14 500 g for 10 min at 5°C. The precipitate was

Protein quantification was carried out according to Bradford (1976). Molecular weight estimation was performed on SDSPAGE according to Laemmli (1970). Oligosaccharide-containing proteins were oxidized with periodate and visualized after staining with Schiff’s reagent according to Leach et al. (1980). Amino acid analysis of the IBP32 was performed after hydrolysis in 6N HCl at 110°C for 22 h in evacuated sealed tubes. The resulting amino acids were reacted with phenylisothiocyanate (PITC) in ethanol: water: triethylamine (7:1:1:1), for 20 min at room temperature, freeze-dried and then dissolved in a small amount of 5 mM sodium phosphate buffer pH 7.4 containing 10% H 3PO4 and 5% acetonitrile. the reaction mixture was applied to a Pico-Tag analysis column (Waters), which was pre-incubated with 0.14 M sodium acetate pH 6.4 containing 0.07% triethylamine and 6% acetonitrile and kept at 40°C. The elution of the PITC amino acids was performed with a gradient of 0 to 34% acetonitrile (60%) in the same buffer formed during 20 min. Protein separation by SDS-PAGE was performed as described earlier. The separated proteins were either stained with Coomassie Brilliant Blue or electroblotted onto a nitrocellulose membrane (Hybond-ECL, Amersham) (Burnette 1981). For immuno-staining the membrane was incubated with a buffer containing a 1:300 dilution of the primary rabbit polyclonal antibody against pistachio male IBP32. After several washes, a secondary goat anti-rabbit conjugated to peroxidase (Jackson Immunoresearch) was added at a 1:1500 dilution. The secondary goat antibody was detected by a peroxidase colour reaction. In cross-reactivity experiments an antidehydrin antiserum directed against a maize dehydrin was used (Close et al. 1993).

Results In a previous investigation (Golan-Goldhirsh 1995), we demonstrated the presence of a 32 kDa protein (IBP32) in an extract of a male inflorescence bud of pistachio. This bud protein constitutes approximately 7.2% of the total proteins of the bud and it disappears during the early stages of microsporogenesis. It was purified to electrophoretic homogeneity (Fig. 2). Analysis of purified IBP32 by SDS-PAGE (Fig. 2) revealed a single protein band of approximately MW

417 Fig. 2 SDS-polyacrylamide gel electrophoresis of female inflorescence bud extract (lane 1), showing the IBP32 and IBP27, and the electrophoretically purified IBP32 (lane 2). Lanes 1 and 2 contained 50 µg and 5 µg proteins, respectively&ig.c:/f

Table 1 Amino acid composition of pistachio inflorescence bud protein (IBP32). Amino acid composition after 22 h hydrolysis in 6 N HCl at 110 0C. The number of amino acid is based on 6 phenylalanine residues per mole. (Glx Glu+Gln and Asx Asp+Asn. Phi hydrophilic: pho hydrophobic, ND not determined)&/tbl.c:&

a

According to Bigelow and Channon (1976)&/tbl.:

32 000, in comparison to a female inflorescence bud extract which contained in addition to the 32 kDa (IBP32), a 27 kDa band (IBP27) which constituted approximately 3.2% of the total proteins of the bud during winter (Fig. 3). The IBP32 and IBP27 contain oligosaccharide moieties, as was detected by PAS staining method for glycoproteins (data not shown). An amino acid analysis of the purified IBP32 protein revealed a content of 284–289 residues per molecule (Table 1). Particularly striking is the high content of glycine and alanine (about 40%) and high hydrophilicity (66% hydrophilic amino acids), calculated according to Bigelow and Channon (1976) (Table 1). The timing of accumulation of the IBP32 is shown in Fig. 3. Proteins of buds collected during the first burst of growth (Fig. 1) were subjected to immunoblotting analysis (Fig. 3), using specific polyclonal antibodies raised against the pistachio male IBP32. Female (lanes 1–3) and male (lanes 4–6) bud proteins were compared at the beginning and end of May (early bud development) and Fig. 3 Immuno detection of IBPs from female (lanes 1–3) and male (lanes 4–6) inflorescence buds of Pistacia vera. A 50 µg protein sample was applied to each lane. lanes 1 and 4 – buds harvested in May 4; lanes 2 and 5 – buds harvested on May 28; lanes 3 and 6 – buds harvested on November 10; Bl – blank sample buffer&ig.c:/f

Amino Acid

Residues/mol

Asxc (phi) d 13 Thr (phi) 7 Ser (phi) 5–6 Glxc (phi) 33 Pro (pho) 11 Gly (phi) 86 Ala (pho) 38 1/2-Cys (phi) 6 Val (pho) 17–18 Met (pho) 5 Ile (pho) 14 Leu (pho) 0 Tyr (phi) 10–11 Phe (pho) 6 His (phi) 6 Lys (phi) 19–20 Trp (pho) NDe Arg (phi) 8–9 Hydrophobicitya 725

in November, at dormancy. IBP32 and IBP27 accumulated during this period to the highest concentration in November (lanes 3 and 6, Fig. 3). The IBPs are expressed throughout the dormant period and disappeared rapidly at the beginning of the second burst of growth (Fig. 1), at about the tetrad stage of microsporogenesis (GolanGoldhirsh 1995). Anti-dehydrin antibodies from maize reacted with IBP32 and IBP27 on Western blots, indicating immunological epitope similarity between the pistachio proteins and maize dehydrins. A comparison of bud proteins of female and male trees, harvested during bud dormancy showed the sexual relatedness of the IBP27 (Fig. 4). None of the eight male trees tested expressed the IBP27 protein, but 26 of 37 female trees did. These proteins were not detected in vegetative buds, leaves or seeds but only in reproductive buds.

Discussion The timing of accumulation of the 32 kDa (IBP32) and the 27 kDa (IBP27) proteins during entry to dormancy indicates a potential role for these proteins either as nitrogen storage material for flower development upon fast growth of the reproductive bud during microsporogenesis in spring, or as protectants of cellular integrity against freezing and dehydration during winter.

418 Fig. 4 Western blot of male (lanes 1–4) and female (lanes 5–8) inflorescence bud extracts, showing the IBP32 and IBP27. Each lane contained 16 µg protein and represents a different tree. Tree’s identification numbers at the Avdat Run-off Farm, according to lane are as follows: lane 1 – 529B; lane 2 – 516B; lane 3 – 515B; lane 4 – 513; lane 5 – 540; lane 6 – 517; lane 7 – 514; lane 8 – 512; Bl – blank sample buffer&ig.c:/f

Dormancy in deciduous trees is a complex developmental process, controlled by genetic, as well as environmental factors. The research of dormancy in deciduous trees and its regulation at the molecular level is at its infancy. The IBP32 and IBP27 of the inflorescence bud were also detected in P. vera bark and other deciduous trees (Golan-Goldhirsh and Shachak 1998). The crossreactivity between anti-dehydrin antibodies from maize with the pistachio proteins, the high glycine content and the high proportion of hydrophilic amino acids in these proteins indicates similarity with the proteins of the dehydrin family. This group of proteins was reported mainly from herbaceous species and only recently from woody plants (Arora et al 1994; Muthalif and Rowland 1994; Wisniewski et al. 1996). In peach, it was shown that a dehydrin-like protein accumulates in winter and disappears in spring (Arora and Wisniewski 1992). It may be hypothesized that these proteins play a role in cold tolerance in deciduous trees during winter. The specific structural properties of these proteins which attribute this functionality are under investigation in our laboratory. The finding that the IBPs are synthesized during early development of the bud (Fig. 1), remain unchanged during the dormancy period, and disappear during flowering – constitutes an intriguing pattern. They may be involved in maintenance of dormancy or, possibly, in sensing an environmental signal for dormancy break. Furthermore, they may serve as cold protectants of the bud during winter and as a nitrogen store for new growth during inflorescence bud sprouting in spring. The close correlation of IBP27 with female buds is intriguing and provides a diagnostic potential. The fact that they accumulate in relatively large quantities makes it less likely that they have a regulatory role; nonetheless, they represent a different developmental pattern in male and female trees and can serve as molecular markers. The question arises whether the IBP27 is an independent gene product, an alternative splicing product of the IBP32 mRNA or a post-translational processing product of IBP32. The cross-reactivity of the IBP32 points towards the second and third alternatives. Characterization of pistachio IBPs at the molecular level and the genetic female sex linkage of IBP27 as well as its relationship to IBP32 is now under investigation in our laboratory.

&p.2:Acknowledgements The technical assistance of Mrs. Ilana Salomon is deeply appreciated. A generous gift by Dr. Timothy J. Close of anti-dehydrin antiserum is greatly appreciated. This research was supported partially by an AID-CDR grant No. DPF5544-G-SS-7013-00 and the Rashi Foundation.

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