A brief note on the study of yieldin,a wall-bound protein that regulates the yield threshold of the cell wall

Yielding of plant-cell walls is defined by the mechanical properties of walls such as their extensibility (Φ for in vivo, φ for in vitro cell wall) and yield threshold (Y for in vivo, y for in vitro cell wall). A protein named yieldin, isolated from the cell wall of growing hypocotyls of Vigna unguiculata L. (cowpea), has been demonstrated to regulate the pH dependency of y in the cell wall of the glycerinated hollow cylinder of the cowpea hypocotyl. This mini-review outlines the process of the discovery of yieldin, and also refers to the progress of studies on yieldin: the molecular cloning of yieldin and its immunolocalization in the etiolated cowpea hypocotyl. Electronic Publication     

The isolation of wall-bound proteins regulating yield threshold tension in glycerinated hollow cylinders of cowpea hypocotyl

To isolate and purify the factor regulating the yield threshold tension (y) through acidification of the cell wall, proteins were extracted from hypocotyls of Vigna unguiculata L. Their effects on the pH‐dependencies of the wall extensibility (φ) and y were examined with reconstitution experiments by incorporating them into the heat‐denatured glycerinated hollow cylinders (GHCs). The wall mechanical properties of the reconstituted GHCs were determined using stress–strain experiments. Only the proteins extracted with 1 kmol m^–3 of NaCl from the wall of elongation region restored the pH‐dependencies of φ and y once extinguished with heat‐denaturation, but proteins extracted from the other cell constituents or from the mature region of hypocotyl affect neither properties. Fractionation of the wall‐bound proteins by a hydrophobic column chromatography showed that the two different fractions affected φ or y independently. The sodium dodecyl sulphate‐polyacrylamide gel electrophoresis showed that the active fraction which restored the pH‐dependency of y still consists of two proteins of 30 and 32 kDa after purification by the sequential fractionation with cation‐exchange and gel filtration. These two proteins were named as ‘yieldin 30’ and ‘32’. Western blotting analysis using the rabbit‐antiserum against the cucumber expansin indicated that the yieldins are independent of cucumber expansin.     

Molecular Cloning of a cDNA Encoding Ribosome Inactivating Protein from Amaranthus viridis and Its Expression in E. coli

In order to isolate a cDNA clone of ribosome inactivating protein (RIP), a cDNA library was constructed in Uni-ZAP XL vector with poly(A) RNA purified from leaves of Amaranthus viridis. To get the probe for screening the library, PCR of phage DNA was conducted using the vector primer and degenerate primer designed from a conserved putative active site of the RIPs. Twenty-six cDNA clones from about 600,000 plaques were isolated, and one of these clones was fully sequenced. It was 1,047 bp and contained an open reading frame encoding 270 amino acids. The deduced amino acid sequence had a putative signal sequence of 17 amino acids and a putative active site (AIQMVAEAARFFKYIE) conserved in other RIPs. E. coli cells expressing A. viridis RIP cDNA did not grow well as compared to control cells, indicating that recombinant A. viridis RIP presumably inactivated E. coli ribosomes. In addition, recombinant A. viridis RIP cDNA produced by E. coli had translation inhibition activity in vitro.     

Natural Infection by Tospoviruses of Cucurbitaceous and Fabaceous Vegetable Crops in India

Natural infection of tospoviruses on three cucurbitaceous (Cucumis sativus, cucumber; Luffa acutangula, ridge gourd; Citrullus lanatus, watermelon) and three fabaceous (Vigna unguiculata, cowpea; Phaseolus vulgaris, French bean; Dolichos lablab, sem) vegetable crops in India was identified on the basis of nucleocapsid protein (NP) gene characteristics. The complete NP gene of the cowpea isolate from Kerala and the sem isolate from Tamil Nadu was 831 nucleotides long, encoding a protein of 276 amino acids. For other Tospovirus isolates from cucumber, French bean, ridge gourd and watermelon, the partial NP gene (291 nt) was sequenced. Comparative NP gene sequence analyses revealed that fabaceous isolates shared maximum identity both at the nucleotide (92–97%) and amino acid (93–97%) levels with the corresponding region of Groundnut bud necrosis virus (GBNV), whereas cucurbitaceous isolates shared maximum identity both at nucleotide (93–99%) and amino acid (95–98%) levels with the corresponding region of Watermelon bud necrosis virus (WBNV), results suggesting that the Tospovirus isolates infecting fabaceous hosts should be regarded as strain of GBNV, whereas those infecting cucurbitaceous hosts as a strain of WBNV. Nucleocapsid protein gene was conserved both in GBNV and WBNV isolates originating from different hosts and locations.     

In vitro plant regeneration via organogenesis of cowpea [Vigna unguiculata (L.) Walp.]

Shoot regeneration via organogenesis was achieved from axenic cowpea [Vigna unguiculata subsp. unguiculata L. (Walp.) Verde.] hypocotyls and cotyledons of advanced breeding lines and varieties. Cotyledons and embryos were excised from green immature pods. The apical parts of the embryos were removed and the hypocotyls were transferred to regeneration media. Cotyledons and hypocotyls were tested on media with gradients of several hormonal and putrescine combinations. Cowpea cotyledons and hypocotyls exhibited a pattern of shoot formation that occurred in three distinct phases. Multiple shoots developed within 45 days from the wounded region of the primary hypocotyl and cotyledons in different media containing a high cytokinin concentration. The induced plant explants were then grown for 20 days in low-intensity light (10 μmol m^–2 s^–1) on the same medium and numerous shoot buds emerged de novo from the upper part of the hypocotyl and the wounded part of the cotyledons. These buds had no apparent vascular connection with the parent tissues. The plant regeneration capability of this procedure was tested with several cowpea genotypes, five of which (83D-442, 86D-1010, 93K-624, Vita 3 and Ife Brown) responded positively with shoot development and were able to form roots and whole plants. Some somaclonal variation was observed. Received: 14 June 1996 / Revision received: 14 December 1996 / Accepted: 25 January 1997     

Development of PCR-based chloroplast DNA markers that characterize domesticated cowpea (Vigna unguiculata ssp. unguiculata var. unguiculata) and highlight its crop-weed complex

In 1992, Vaillancourt and Weeden discovered a very important mutation for studying cowpea evolution and domestication. A loss of a BamHI restriction site in chloroplast DNA characterized all domesticated accessions and a few wild (Vigna unguiculata ssp. unguiculata var. spontanea) accessions. In order to screen a larger number of accessions, primers were designed to check this mutation using PCR RFLP or direct PCR methods. Using these new primers, 54 domesticated cowpea accessions and 130 accessions from the wild progenitor were screened. The absence of haplotype 0 was confirmed within domesticated accessions, including primitive landraces from cultivar-groups Biflora and Textilis, suggesting that this mutation occurred prior to domestication. However, 40 var. spontanea accessions distributed from Senegal to Tanzania and South Africa showed haplotype 1. Whereas this marker could not be used to identify a precise center of origin, it did highlight the widely distributed cowpea crop-weed complex. Its very high frequency in West Africa could be interpreted as a result of either genetic swamping of the wild/weedy gene pool by the domesticated cowpea gene pool or as the result of domestication by ethnic groups focusing primarily on cowpea as fodder.     

CHLOROPLAST DNA POLYMORPHISM SUGGESTS NIGERIAN CENTER OF DOMESTICATION FOR THE COWPEA,VIGNA UNGUICULATA (LEGUMINOSAE)

Twenty-one independent chloroplast DNA polymorphisms were identified in Vigna unguiculata defining 19 different chloroplast DNA molecules (plastome types). Two plastome types, differing by a single character, were found among 32 accessions of cultivated cowpea (Vigna unguiculata ssp. unguiculata). Eighteen different plastome types were found among 26 accessions of wild cowpea (V. unguiculata ssp. dekindtiana). The very low level of chloroplast DNA diversity found in cultivated accessions relative to wild cowpea suggests that 1) the domesticated form was derived from a narrow selection of the wild germplasm and 2) chloroplast gene flow between wild and cultivated types has been very limited. Cladistic analysis of the cpDNA data generated a robust tree completely lacking homoplasy. Three wild accessions from Nigeria possessed a plastome type indistinguishable from one present in cultivated accessions, suggesting that Nigeria represents one center of domestication of the cowpea. The other plastome type within the cultivated germplasm was not found among wild accessions.     

Isolation of a cDNA fragment coding for Chlamydomonas reinhardtii ferredoxin and expression of the recombinant protein in Escherichia coli

A cDNA clone coding for mature C. reinhardtii ferredoxin has been isolated from a cDNA library using PCR and two oligonucleotide primers based on the N- and C-termini of the protein's amino acid sequence. The nucleotidic sequence of the PCR fragment (299 bp) agreed well with the amino acid sequence since a single conservative substitution (Thr-7 to Ser) could be deduced. The PCR fragment was inserted into the expression vector pTrc 99A, using the incorporated NcoI and BamHI restriction sites and the construction used to transform E. coli (DH5α F′). After subsequent large scale expression and purification of the recombinant protein, biochemical and biophysical analysis have indicated that the product isolated from E. coli is homologous to native ferredoxin isolated from green algae.     

Molecular cloning and characterization of a mannose-binding lectin gene from <Emphasis Type="Italic">Pinellia cordata</Emphasis>

Using RNA extracted from Pinellia cordata young leaves and primers designed according to the conserved regions of Araceae lectins, the full-length cDNA of Pinellia cordata agglutinin (PCL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of pcl was 1,182 bp and contained a 768 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acids. Through comparative analysis of pcl gene and its deduced amino acid sequence with those of other Araceae species, it was found that pcl encoded a precursor lectin with signal peptide. PCL is a mannose-binding lectin with three mannose-binding sites. Semi-quantitative RT-PCR analysis revealed that pcl is expressed in all tested tissues including leaf, stem and bulbil, but with the highest expression in bulbil. PCL protein was successfully expressed in Escherichia coli with the molecular weight expected.     

Implications of the galactosidase activity of yieldin in the regulatory mechanism of yield threshold that is fundamental to cell wall extension

To understand the action mechanism of yieldin (YLD) on the regulation of the yield threshold (Y ), one of the critical parameters of cell wall extension, YLD was extracted from the cell walls of cowpea (Vigna unguiculata L.) hypocotyls and the hemagglutinin activity (HA) as well as the glycosidase activity of the protein was measured. Sedimentation assays using trypsinated rabbit erythrocytes showed that YLD possessed HA at pH 7. The digestion assays using 4‐nitrophenyl (p NP) glycopyranosides as artificial substrates showed that YLD liberated galactose residues from p NP alpha‐d ‐galactopyranoside mainly at pH 4.0, i.e. the pH level where Y was decreased at most. These results show that YLD is a bifunctional protein that switches between the HA and the galactosidase activities depending on the surrounding pH. Since D‐galactose at concentration of 0.03 g l^?1 perfectly inhibited the HA, YLD was suggested to associate with galactose residues. However, the galactose application ten times concentrated was necessary to inhibit both the galactosidase activity of YLD and the acid‐induced shift of Y regulated by YLD. In addition, the specific inhibitor of alpha‐d ‐galactosidase (deoxygalactonojirimycin) inhibited both the galactosidase activity of YLD and the shift of Y at the same concentration, but not the HA. On the basis of these results, it is suggested the galactosidase activity of YLD plays a central role in the mechanism of Y ‐regulation at acidic pH.     

Cloning and expression of UDP-glucose: flavonoid 7-O-glucosyltransferase from hairy root cultures of Scutellaria baicalensis

 A cDNA encoding UDP-glucose: baicalein 7-O-glucosyltransferase (UBGT) was isolated from a cDNA library from hairy root cultures of Scutellaria baicalensis Georgi probed with a partial-length cDNA clone of a UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) from grape (Vitis vinifera L.). The heterologous probe contained a glucosyltransferase consensus amino acid sequence which was also present in the Scutellaria cDNA clones. The complete nucleotide sequence of the 1688-bp cDNA insert was determined and the deduced amino acid sequences are presented. The nucleotide sequence analysis of UBGT revealed an open reading frame encoding a polypeptide of 476 amino acids with a calculated molecular mass of 53 094 Da. The reaction product for baicalein and UDP-glucose catalyzed by recombinant UBGT in Escherichia coli was identified as authentic baicalein 7-O-glucoside using high-performance liquid chromatography and proton nuclear magnetic resonance spectroscopy. The enzyme activities of recombinant UBGT expressed in  E. coli were also detected towards flavonoids such as baicalein, wogonin, apigenin, scutellarein, 7,4′-dihydroxyflavone and kaempferol, and phenolic compounds. The accumulation of UBGT mRNA in hairy roots was in response to wounding or salicylic acid treatments. Received: 8 September 1999 / Accepted: 4 October 1999     

cDNA cloning and recombinant expression of the general odorant binding protein II from Spodoptera litura

A cDNA encoding the general odorant binding protein II (GOBP II) was isolated from the antennae of Spodoptera litura (SlGOBP II, GenBank Accession No. EU086371) by homologous cloning and rapid amplification of cDNA ends (RACE). Sequencing and structural analyses revealed that the open reading frame (ORF) of SlGOBP II was 489 bp, encoding 162 amino acids with a predicted MW of 18.2 kD and pI of 5.72. SlGOPB II shared typical structural features of odorant binding proteins with other insects, including the six conservative cysteine residues. The deduced amino acid sequence of SlGOPB II shared significant identity with the GOBP II from S. frugiperda and S. exigua. RT-PCR and Northern blot analyses showed that SlGOBP II was specifically expressed in the antennae. cDNA encoding SlGOBP II was constructed into the pET-32a vector and the recombinant protein was highly expressed in Es-cherichia coli BL21 (DE3) after induction with IPTG. SDS electrophoresis and Western blot analysis confirmed the molecular weight of the recombinant SIGOBPII i.e, 32 kD, which has a 6×His tag at the N-terminus. The recombinant SlGOBP II was purified by single-step Ni-NTA affinity chromatography and used to raise antiserum in rabbits. ELISA showed that the titer of antiserum was 1︰12800, while Western blot analysis showed that the recombinant SlGOBP II was recognized as anti-SlGOBP II an-tiserum.     

Protein and free amino acids in field-grown cowpea seeds as affected by water stress at various growth stages

Summary This study was undertaken to evaluate water stress effects during vegetative, flowering, and podfilling stages of cowpea plants (Vigna unguiculata L.) grown under natural field conditions in southern California on seed yield and protein and free amino acid content of the cowpea seeds. The lowest concentration of N was found in the seeds of the control treatment plants while the seed yield from these treatments was the highest as compared with the N concentration and yield of seeds from plants subjected to water stress during flowering and podfilling stages. The concentration of N in the seeds was inversely related to the seed dry weight yield. Protein arginine,-threonine,-serine,-cystine,-valine,-methionine, and-isoleucine were significantly affected by water stress at the three growth stages. There was no consistent pattern in the effect of water stress on the individual amino acids. The sum of protein amino acids in the cowpea seeds was not significantly influenced by the various treatments since some of the protein amino acids increased and others decreased producing an averaging effect on the figures comprising the sums of the amino acids. Water stress during the flowering and pod-filling stages increased the free amino acid pool, and at the same time, inhibited incorporation of the amino acids into the protein chain-thus lowering the protein amino acid fraction simultaneously. With the exception of methionine plus cystine, the essential amino acids in the seeds were present at concentrations equal to or greater than recommended by the World Health Organization and FAO. It is of particular importance to note that the concentration of lysine in the cowpeas was substantially higher than that found in wheat grain. It is also important to note that the amount of essential amino acids per gram of protein was not measurably affected by the water stress treatments during any of the growth stages.     

Chilling-induced changes of vacuolar proton pumps in hypocotyls of <Emphasis Type="Italic">Vigna unguiculata</Emphasis>

Vigna unguiculata (cowpea) is a legume adapted to high temperatures and is sensitive to low temperatures. Temperature is one of the limiting factors of growth and yield for many crops but its effect on cowpea metabolism is not known. We investigated the effect of chilling on activity of vacuolar proton pumps (V-ATPase and V-PPase) and their protein content in tonoplast vesicles of cowpea hypocotyls. Seedlings grown for 7 days at 10 or 4°C were used for experiments. Chilling treatment at 10 or 4°C markedly suppressed growth of cowpea seedlings. Following chilling at 10 and 4°C, activity of both proton pumps and the relative amount of V-PPase and subunit A of V-ATPase were significantly increased. Both substrate hydrolysis and H⁺ transport activities of V-PPase remained at relatively high levels during chilling treatment. For V-ATPase, treatment at 10°C for 6 days increased the ATP hydrolysis activity. However, the H⁺ transport activity of the enzyme was increased when treated for 4 days but was markedly decreased when treated for 6 days. Our results provide evidence for different regulation for these vacuolar proton pumps, indicating that V-PPase is the more stable proton pump throughout chilling stress.     

Preparation and properties of mitochondria from cowpea nodules

Mitochondria were isolated from nodules of cowpea (Vigna unguiculata (L). Walp.) and purified on a Percoll gradient. They were only slightly contaminated by bacteroids (an average of 3.5%), and had low lipoxygenase activity. Compared to mitochondria from hypocotyls the nodule mitochondria had similar O₂ uptake rates and respiratory control ratios. The ADP/O ratios for both preparations were 1.4 to 1.7 and 2.3 to 2.6 with succinate and malate, respectively. Whereas mitochondria isolated from etiolated cowpea hypocotyls had 14 to 18% of their respiration insensitive to KCN, the respiration of nodule mitochondria was completely inhibited by KCN. Enzyme activities of nodule mitochondria were similar to those found in hypocotyl mitochondria, except for NAD⁺-malic enzyme which was 12-fold lower in the mitochondria from nodules.     

Porcine pyridoxal kinase: c-DNA cloning, expression and confirmation of its primary sequence

Porcine brain pyridoxal kinase has been cloned. A 1.2 kilo-based cDNA with a 966-base pair open reading frame was determined from a porcine brain cortex cDNA library using PCR technique. The DNA sequence was shown to encode a protein of 322 amino acid residues with a molecular mass of 35.4 kDa. The amino acid sequence deduced from the nucleotide sequence of the cDNA was shown to match the partial primary sequence of pyridoxal kinase. Expression of the cloned cDNA in E. coli has produced a protein which displays both pyridoxal kinase activity and immunoreactivity with monoclonal antibodies raised against natural enzyme from porcine brain. With respect to the physical properties, it is shown that the recombinant protein exhibits identical kinetic parameters with the pure enzyme from porcine brain. Although the primary sequence of porcine pyridoxal kinase has been shown to share 87% homology with the human enzyme, we have shown that the porcine enzyme carries an extra peptide of ten amino acid residues at the N-terminal domain.     

<Emphasis Type="Italic">Aspergillus niger</Emphasis> lipase: gene cloning,over-expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> and in vitro refolding

From the N-terminal amino acid sequence of the lipase from Aspergillus niger F044, a potential homologous gene A84689 to the lipanl (the gene encoding the lipase from Aspergillus niger F044) was identified. A pair of primers were designed according to the nucleotide sequence of A84689, and the lipanl was cloned by PCR. Nucleotide sequencing revealed that the lipanl has an ORF of 1,044 bp, containing three introns. The deduced amino acid sequence corresponds to 297 amino acid residues. The cloned cDNA fragment encoding the mature lipase from Aspergillus niger F044 was over-expressed in Escherichia coli BL21(De3) and the recombinant protein was refolded in vitro by dilution followed by DEAE Sepharose Fast Flow chromatography.