Showing papers by "Natasha V. Raikhel published in 1989"

Cloning and Characterization of Root-Specific Barley Lectin

Abstract: Cereal lectins are a class of biochemically and antigenically related proteins localized in a tissue-specific manner in embryos and adult plants. To study the specificity of lectin expression, a barley (Hordeum vulgare L.) embryo cDNA library was constructed and a clone (BLc3) for barley lectin was isolated. BLc3 is 972 nucleotides long and includes an open reading frame of 212 amino acids. The deduced amino acid sequence contains a putative signal peptide of 26 amino acid residues followed by a 186 amino acid polypeptide. This polypeptide has 95% sequence identity to the antigenically indistinguishable wheat germ agglutinin isolectin-B (WGA-B) suggesting that BLc3 encodes barley lectin. Further evidence that BLc3 encodes barley lectin was obtained by immunoprecipitation of the in vitro translation products of BLc3 RNA transcripts and barley embryo poly(A+) RNA. In situ hybridizations with BLc3 showed that barley lectin gene expression is confined to the outermost cell layers of both embryonic and adult root tips. On Northern blots, BLc3 hybridizes to a 1.0 kilobyte mRNA in poly(A+) RNA from both embryos and root tips. We suggest, on the basis of immunoblot experiments, that barley lectin is synthesized as a glycosylated precursor and processed by removal of a portion of the carboxyl terminus including the single N-linked glycosylation site.

Stress-Induced Accumulation of Wheat Germ Agglutinin and Abscisic Acid in Roots of Wheat Seedlings

Abstract: Wheat germ agglutinin (WGA) levels in roots of 2-day-old wheat seedlings increased up to three-fold when stressed by air-drying. Similar results were obtained when seedling roots were incubated either in 0.5 molar mannitol or 180 grams per liter polyethylene glycol 6000, with a peak level of WGA after 5 hours of stress. Longer periods of osmotic treatment resulted in a gradual decline of WGA in the roots. Since excised wheat roots incorporate more [35S]cysteine into WGA under stress conditions, the observed increase of lectin levels is due to de novo synthesis. Measurement of abscisic acid (ABA) levels in roots of control and stressed seedlings indicated a 10-fold increase upon air-drying. Similarly, a five- and seven-fold increase of ABA content of seedling roots was found after 2 hours of osmotic stress by polyethylene glycol 6000 and mannitol, respectively. Finally, the stress-induced increase of WGA in wheat roots could be inhibited by growing seedlings in the presence of fluridone, an inhibitor of ABA synthesis. These results indicate that roots of water-stressed wheat seedlings (a) contain more WGA as a result of an increased de novo synthesis of this lectin, and (b) exhibit higher ABA levels. The stress-induced increase of lectin accumulation seems to be under control of ABA.

Expression of rice lectin is governed by two temporally and spatially regulated mRNAs in developing embryos.

Abstract: Two cDNA clones encoding rice lectin have been isolated and characterized to investigate the expression of rice lectin at the molecular and cellular levels. The two cDNA clones code for an identical 23-kilodalton protein which is processed to the mature polypeptide of 18 kilodaltons by co-translational cleavage of a 2.6-kilodalton signal sequence and selective removal of a 2.7-kilodalton COOH-terminal peptide which contains a potential N-linked glycosylation site. In addition, the mature 18-kilodalton lectin is post-translationally cleaved between residues 94 and 95 to yield polypeptides of 10 kilodaltons and 8 kilodaltons, corresponding to the NH2- and COOH-terminal portions of the mature subunit, respectively. RNA gel blot analysis established that rice lectin is encoded by two mRNA transcripts (0.9 kilobase and 1.1 kilobase). On DNA gel blots, the rice lectin cDNAs hybridize specifically to a single restriction fragment. In situ hybridization showed localization of the 1.1-kilobase rice lectin mRNA in root caps and specific cell layers of the radicle, coleorhiza, scutellum, and coleoptile. RNA gel blot analysis demonstrated that both the 0.9-kilobase and 1.1-kilobase mRNAs are present in developing rice embryos. The two lectin mRNAs are differentially expressed temporally such that the 1.1-kilobase lectin mRNA accumulates to levels twofold higher than the 0.9-kilobase mRNA.

Nucleotide sequences of cDNA clones encoding wheat germ agglutinin isolectins A and D.

Abstract: In hexaploid wheat (Triticum aestivum L.), the N-acetylglucosamine-binding lectin wheat germ agglutinin (WGA) consists of three unique isolectins (A, B and D) encoded by their respective diploid genomes [2]. Previous observations suggesting that the genes for individual isolectins might be expressed in a differential or tissue-specific manner [3] prompted us to isolate eDNA clones corresponding to each of the three WGA isolectins. We previously reported the sequence of a 927 nucleotide eDNA (WGA-B) encoding WGA isolectin B [4]. Using the insert from WGA-B we probed a 2gtl0 cDNA library prepared with poly(A) + RNA from developing embryos of Triticum aestivum L. cv. Marshall, and isolated two full-length cDNA clones encoding isolectins A and D (WGA-A and WGA-D, respectively). Figure 1 gives the complete nucleotide sequences of WGA-A and WGA-D as determined by sequencing overlapping, single-stranded fragments [1]. WGA-A and WGA-D both contain short 5' untranslated regions (21 and 4 nucleotides, respectively) followed by an open reading frame of 212 (WGA-A) or 213 (WGA-D) amino acids. These open reading frames are divided into three regions: i)a putative signal peptide (26 amino acids, WGA-A; 27 amino acids, WGA-D); ii) 171 amino acids with 100~o identity with the published amino acid sequences of either WGA isolectin A (WGA-A) or WGA isolectin D (WGA-D) [6]; and iii)a 15 amino acid carboxyl-terminal propeptide containing an N-linked glycosylation site as described for WGA-B [4 ]. The nucleotide sequence of WGA-A possesses 94.0 ~o identity with WGA-D (94.8 ~o in the coding region) and 91.8~o identity with WGA-B (93.7~o in the coding region), while WGA-D possesses 91.5 ~o identity with WGA-B (93.0~o in the coding region). The high degree of similarity observed in the 3' untranslated regions was surprising, as earlier data predicted that the WGA isolectin genes would have enough differences to produce isolectin-specific probes [5]. The cDNA sequences presented here failed to fulfill that function as WGA-A, WGA-B and WGA-D cross-hybridized under stringent criteria and hybridized in an identical fashion to Southern blots of genomic DNA from diploid wheats (not shown).

Sequence variability in three wheat germ agglutinin isolectins: Products of multiple genes in polyploid wheat

Abstract: Three highly homologous wheat germ isolectins (95–97%) are distinct gene products in hexaploid wheat. The amino acid sequences of two of these [wheat germ agglutinin 1 (WGA1) and 2 (WGA2)] are compared with sequence date derived from a complementary DNA (cDNA) clone for the third isolection (WGA3). This comparison includes three corrections to earlier amino acid sequences data of both WGA1 and WGA2 at positions 109 (from Ser to Phe), 134 (from Gly to Lys), and 150 (from Gly to Trp). These reassignments are based on new results from crystal structure refinement and amino acid sequence data of WGA1, as well as the recently determined nucleotide sequence of WGA3. In addition, the C-terminal residue of WGA1 has been revised to Gly 171 and now differs from WGA2 (Ala 171). Four other positions, Asn9, Ala53, Gly119, and Ser 123, at which WGA1 and WGA2 are identical but differ from the DNA sequence of WGA3, were also reinvestigated by amino acid sequencing techniques and confirmed.

In situ RNA hybridization in plant tissues

Abstract: In situ hybridization is the most direct way of examining the modulation of gene expression during development at the individual cell level. This technique has been used primarily in animal tissues to determine the location of a particular mRNA or to map chromosomal positions of cloned DNA [2, 3, 9, 10, 24, 30]. This approach has also been used as a diagnostic tool for the detection of cells infected with viruses [6, 12, 19]. More recently, in situ hybridization has been applied to the localization of specific RNAs in plant tissues.

Production of an antibody specific for the propeptide of wheat germ agglutinin.

Abstract: Wheat germ agglutinin (WGA) is synthesized as a proprotein with a glycosylated, 15 amino acid, carboxyl-terminal propeptide. This glycopeptide is cleaved from pro-WGA to produce the mature lectin during the transport of WGA to the protein bodies/vacuoles. To study the posttranslational modification of WGA, it would be useful to be able to differentiate between pro-WGA and mature WGA. Therefore, a peptide corresponding to the propeptide of WGA was synthesized (WGA-B 172-186), and an antiserum was raised in rabbits (anti-WGA-B 172-186). Anti-WGA-B 172-186 reacted with pure WGA-B 172-186 and pro-WGA in ELISA. Anti-WGA-B 172-186 was also specific for and readily differentiated between pro-WGA and mature WGA on Western blots. This provided an assay to monitor pro-WGA on Western blots before and after endo-β-N-acetylglucosaminidase H digestion. Using this assay, direct evidence was obtained that the oligosaccharide of pro-WGA is of the high-mannose type.

Stress-induced Accumulation ofWheatGermAgglutinin and Abscisic AcidinRootsofWheatSeedlings1

Abstract: Wheatgermagglutinin (WGA)levels inroots of2-day-old wheat seedlings increased uptothree-fold whenstressed byair-drying. Similar results wereobtained whenseedling roots wereincubated either in0.5molarmannitol or180gramsperliter polyethylene glycol 6000, withapeaklevel ofWGA after 5hoursofstress. Longer periods ofosmotic treatment resulted inagradual decline ofWGAintheroots. Since excised wheatroots incorporate more [35S]cysteine intoWGA understress conditions, theobserved increase oflectin levels isduetodenovosynthesis. Measurementofabscisic acid(ABA)levels inroots ofcontrol andstressed seedlings indicated a10-fold increase uponair-drying. Similarly, afive- andseven-fold increase ofABAcontent ofseedling roots wasfoundafter 2hours ofosmotic stress bypolyethylene glycol 6000andmannitol, respectively. Finally, thestress-induced increaseofWGA inwheatrootscouldbeinhibited bygrowing seedlings inthepresence offluridone, aninhibitor ofABAsynthesis. Theseresults indicate that roots ofwater-stressed wheat seedlings (a)contain moreWGA asaresult ofanincreased de novosynthesis ofthis lectin, and(b)exhibit higher ABAlevels. Thestress-induced increase oflectin accumulation seemstobe under control ofABA. havebeenmanyinvestigations onstress-induced ABAaccumulation inleaves, fewstudies haveexamined endogenous ABAlevels inplant roots. Thispaper reports enhanced ABA levels inroots ofwater-stressed wheat seedlings. Since ABA reportedly stimulates WGA synthesis inwheat roots (16), we further describe (a) theincrease ofWGA content andsynthesis inwheatseedling roots asaresult ofdrought andosmotic stress, and(b) theinhibition ofthis stress-induced increase by fluridone, aninhibitor ofABAsynthesis (2, 4,5,14).