Nectarin I is a novel,soluble germin-like protein expressed in the nectar of Nicotiana sp.

We have identified a limited number of proteins secreted into the nectar of tobacco plants. Nectarin I is the most highly expressed nectar protein and has a monomer molecular mass of 29 kDa. The other major nectar proteins are expressed at lower levels and have monomer molecular masses of 41, 54, and 65 kDa respectively. Nectarin I was purified and antiserum was raised against the protein. Under nondenaturing conditions, Nectarin I has an apparent molecular mass of >120 kDa. The expression of Nectarin I was restricted to nectary tissues and to a much lower level in the ovary. No Nectarin I was found in petals, stems, leaves, or roots or other floral tissues. The expression of Nectarin I was also developmentally regulated. It is expressed in nectary tissues only while nectar is being actively secreted. Subsequently, the N-terminus of purified Nectarin I was sequenced. Sequence identity showed Nectarin I is related to wheat germin. Although hydrogen peroxide is readily detectable in tobacco floral nectar, we were unable to demonstrate any oxalate oxidase activity for Nectarin I. A partial cDNA encoding the mature Nectarin I N-terminus was isolated and used to probe a Nicotiana plumbaginifolia genomic library. The Nectarin I gene was isolated and the translated sequence was consistent with both N-terminal and internal cyanogen bromide-derived amino acid sequence. The gene contains a single 386 nt intron and encodes a mature protein of 197 amino acids.     

Alfalfa Root Nodule Carbon Dioxide Fixation : III. Immunological Studies of Nodule Phosphoenolpyruvate Carboxylase

Antiserum was prepared in rabbits against purified alfalfa (Medicago sativa L.) nodule phosphoenolpyruvate carboxylase (PEPC). Immunotitration assays revealed that the antiserum recognized the enzyme from alfalfa nodules, uninoculated alfalfa roots, and from soybean nodules. Tandem-crossed immunoelectrophoresis showed that the PEPC protein from alfalfa roots and nodules was immunologically indistinguishable. The 101 kilodalton polypeptide subunit of alfalfa nodule PEPC was identified on Western blots. The PEPC polypeptide was detected in low quantities in young alfalfa roots and nodules but was present at increased levels in mature nodules. Senescent nodules appeared to contain a reduced amount of the PEPC polypeptide. PEPC was also detected by western blot in some plant- and bacterially-conditioned ineffective alfalfa nodules but was not detected in bacteroids isolated from effective nodules. Alfalfa nodule PEPC is constitutively expressed in low levels in roots. In nodules, expression of PEPC polypeptide increases several-fold, resulting in increased PEPC activity. Antiserum prepared against the C₄ PEPC from maize leaves recognized the PEPC enzyme in all legume nodules and roots tested, while the antiserum prepared against alfalfa nodule PEPC also recognized the leaf PEPC of several C₄ plant species. Neither antiserum reacted strongly with any C₃ leaf proteins. The molecular weight of the PEPC polypeptide from C₄ leaves and legume nodules appears to be similar.     

Association of caffeoyl coenzyme A 3-O-methyltransferase expression with lignifying tissues in several dicot plants.

Caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) was previously shown to be associated with lignification in both in vitro tracheary elements (TEs) and organs of zinnia (Zinnia elegans). However, it is not known whether this is a general pattern in dicot plants. To address this question, polyclonal antibodies against zinnia recombinant CCoAOMT fusion protein were raiseed and used for immunolocalization in several dicot plants. The antibodies predominantly recognized a protein band with a molecular mass of 28 kD on western analysis of tissue extracts from zinnia, forsythia (Forsythia suspensa), tobacco (Nicotiana tabacum), alfalfa (Medicago sativa), and soybean (Glycine max). Western analyses showed that the accumulation of CCoAOMT protein was closely correlated with lignification in in vitro TEs of zinnia. Immunolocalization results showed that CCoAOMT was localized in developing TEs of young zinnia stems and in TEs, xylem fibers, and phloem fibers of old stems. CCoAOMT was also found to be specifically associated with all lignifying tissues, including TEs, xylem fibers, and phloem fibers in stems of forsythia, tobacco, alfalfa, soybean, and tomato (Lycopersicon esculentum). The presence of CCoAOMT was evident in xylem ray parenchyma cells of forsythia, tobacco, and tomato. In forsythia and alfalfa, pith parenchyma cells next to the vascular cylinder were lignified. Accordingly, marked accumulation of CCoAOMT in these cells was observed. Taken together, these results showed a close association of CCoAOMT expression with lignification in dicot plants. This supports the hypothesis that the CCoAOMT-mediated methylation branch is a general one in lignin biosynthesis during normal growth and development in dicot plants.     

A mutation in Thermosensitive Male Sterile 1, encoding a heat shock protein with DnaJ and PDI domains, leads to thermosensitive gametophytic male sterility in Arabidopsis

In most flowering plant species, pollination and fertilization occur during the hot summer, so plants must have evolved a mechanism that ensures normal growth of their pollen tubes at high temperatures. Despite its importance to plant reproduction, little is known about the molecular basis of thermotolerance in pollen tubes. Here we report the identification and characterization of a novel Arabidopsis gene, THERMOSENSITIVE MALE STERILE 1 ( TMS1 ), which plays an important role in thermotolerance of pollen tubes. TMS1 encodes a Hsp40-homologous protein with a DnaJ domain and an a_ERdj5_C domain found in protein disulfide isomerases (PDI). Purified TMS1 expressed in Escherichia coli (BL21 DE3) had the reductive activity of PDI. TMS1 was expressed in pollen grains, pollen tubes and other vegetative tissues, including leaves, stems and roots. Heat shock treatment at 37°C increased its expression levels in growing pollen tubes as well as in vegetative tissues. A knockout mutation in TMS1 grown at 30°C had greatly retarded pollen tube growth in the transmitting tract, resulting in a significant reduction in male fertility. Our study suggests that TMS1 is required for thermotolerance of pollen tubes in Arabidopsis, possibly by functioning as a co-molecular chaperone.     

Specificity of expression of the GUS reporter gene (uidA) driven by the tobacco ASA2 promoter in soybean plants and tissue cultures

Twelve independent lines were transformed by particle bombardment of soybean embryogenic suspension cultures with the tobacco anthranilate synthase (ASA2) promoter driving the uidA (beta-glucuronidase, GUS) reporter gene. ASA2 appears to be expressed in a tissue culture specific manner in tobacco (Song H-S, Brotherton JE, Gonzales RA, Widholm JM. Tissue culture specific expression of a naturally occurring tobacco feedback-insensitive anthranilate synthase. Plant Physiol 1998;117:533-43). The transgenic lines also contained the hygromycin phosphotransferase (hpt) gene and were selected using hygromycin. All the selected cultures or the embryos that were induced from these cultures expressed GUS measured histochemically. However, no histochemical GUS expression could be found in leaves, stems, roots, pods and root nodules of the plants formed from the embryos and their progeny. Pollen from some of the plants and immature and mature seeds and embryogenic cultures initiated from immature cotyledons did show GUS activity. Quantitative 4-methylumbelliferyl-glucuronide (MUG) assays of the GUS activity in various tissues showed that all with observable histochemical GUS activity contained easily measurable activities and leaves and stems that showed no observable histochemical GUS staining did contain very low but measurable MUG activity above that of the untransformed control but orders of magnitude lower than the constitutive 35S-uidA controls used. Low but clearly above background levels of boiling sensitive GUS activity could be observed in the untransformed control immature seeds and embryogenic cultures using the MUG assay. Thus in soybean the ASA2 promoter drives readily observable GUS expression in tissue cultures, pollen and seeds, with only extremely low levels seen in vegetative tissues of the plants. The ASA2 driven expression seen in mature seed was, however, much lower than that seen with the constitutive 35S promoter; less than 2% in seed coats and less than 0.13% in cotyledons and embryo axes. The predominate tissue culture specific expression pattern of the ASA2 promoter may be useful for genetic transformation of crops.     

Distribution of two isoforms of NADP-dependent isocitrate dehydrogenase in soybean (Glycine max)

Two different cDNAs that encode NADP-specific isocitrate dehydrogenase (NADP-IDH) isozymes of soybean (Glycine max) were characterized. The nucleotide sequences of the coding regions of these cDNAs have 74% identity to each other and give predicted amino acid sequences that have 83% identity to each other. Using PCR techniques, their coding regions were subcloned into a protein overexpression vector, pQE32, to yield pIDH4 and pIDH1, respectively. Both IDH4 and IDH1 enzymes were expressed in Escherichia coli as catalytically active His6 tagged proteins, purified to homogeneity by affinity chromatography on nickel chelate resin and rabbit polyclonal antibodies to each were generated. Surprisingly, antiserum to IDH4 did not react with IDH1 protein and IDH1 antiserum reacted only very weakly with IDH4 protein. IDH4 antibody reacts with a protein of expected molecular weight in cotyledon, young leaf, young root, mature root and nodules but the reaction with mature leaf tissue was low compared to other tissues. Western blot results show that IDH1 was not expressed in young roots but a protein that reacts with the IDH1 antibody was highly expressed in leaves, showing that there was tissue-specific accumulation of NADP-IDH isozymes in soybean.     

管氏肿腿蜂雌性抚育中幼虫转移行为的启动和节律

为深入理解肿腿蜂雌性抚育的行为学特征, 在室内连续观察了雌性管氏肿腿蜂Sclerodermus guani对子代幼虫的转移行为, 旨在明确雌蜂在子代蜂幼虫发育到哪一阶段时启动转移行为, 以及幼虫转移行为是否有节律。以黄粉虫Tenebrio militor蛹期在24 h内的蛹体为寄主, 根据子代蜂幼虫发育进程将其划分为低龄幼虫（1-2龄）、 高龄幼虫（3-4龄）、 老熟幼虫(自然脱落)和吐丝幼虫（开始吐丝结茧）等4个时期, 采取人工剥离（早期幼虫）或自然脱离（晚期幼虫）的方法处理子代蜂幼虫, 观察雌蜂对所表现出的行为反应; 然后以子代蜂高龄幼虫为对象, 连续观察雌蜂的30次幼虫转移行为过程。结果表明： 雌蜂对所有发育时期子代蜂幼虫均用触角拍打进行探测; 但不转移低龄幼虫, 只转移其他阶段幼虫, 转移老熟幼虫和吐丝幼虫的瞬间概率分别是转移高龄幼虫的4.09倍和7.69倍。雌蜂转移高龄、 老熟和吐丝幼虫的比例分别为96%, 100%和100%, 没有显著差异（P≥0.05）; 对高龄幼虫、 老熟幼虫和吐丝幼虫转移耗时平均分别为27.96, 34.04和32.49 s, 没有显著差异（P≥0.05）; 平均转移距离依次为4.19, 7.18和 9.43 mm, 对吐丝幼虫的转移距离显著大于高龄幼虫（P<0.05）, 但在高龄和老熟幼虫之间没有显著差异（P≥0.05）。对雌蜂连续30次幼虫转移行为的趋势和节律分析表明： 幼虫转移前探测的幼虫数总体上随幼虫转移次序增加而减少, 在间隔1次和2次之间存在显著自相关, 幼虫转移耗时在间隔1次之间存在显著自相关, 但幼虫转移距离未表现出明显的节律。本研究结果说明, 管氏肿腿蜂雌性抚育中的幼虫转移行为只在子代蜂幼虫发育到较高龄期时启动, 且幼虫转移中的某些行为特征具有节律性。     

Plant endoplasmin supports the protein secretory pathway and has a role in proliferating tissues

Endoplasmin is a molecular chaperone of the heat-shock protein 90 class located in the endoplasmic reticulum and its activity is poorly characterized in plants. We assessed the ability of endoplasmin to alleviate stress via its transient overexpression in tobacco protoplasts treated with tunicamycin, an inhibitor of glycosylation and inducer of the unfolded protein response (UPR). Endoplasmin supported the secretion of a model secretory protein but was less effective than BiP, the endoplasmic reticulum member of the heat-shock protein 70 family. Consistently, immunoprecipitation experiments with in vivo radioactively labelled proteins using an antiserum prepared against Arabidopsis endoplasmin showed that a much smaller number of newly synthesized polypeptides associated with endoplasmin than with BiP. Synthesis of endoplasmin was enhanced by UPR inducers in tobacco seedlings but not protoplasts. As BiP synthesis was induced in both systems, we conclude that the UPR acts differently, at least in part, on the expression of the two chaperones. Endoplasmin was not detectable in extracts of leaves and stems of the Arabidopsis endoplasmin T-DNA insertion mutant shepherd . However, the chaperone is present, albeit at low levels, in shepherd mutant callus, mature roots and tunicamycin-treated seedlings, demonstrating that the mutation is leaky. Reduced endoplasmin in the shepherd mutant has no effect on BiP protein levels in callus or mature roots, leaves and stems, but is compensated by increased BiP in seedlings. This increase occurs in proliferating rather than expanding leaf cells, indicating an important role for endoplasmin in proliferating plant tissues.     

The lysine-rich arabinogalactan-protein subfamily in Arabidopsis: gene expression, glycoprotein purification and biochemical characterization

AtAGP17, AtAGP18 and AtAGP19 are homologous genes encoding three putative glycosylphosphatidylinositol (GPI)-anchored classical arabinogalactan-proteins (AGPs) in Arabidopsis. They are distinguished from other AGPs by a short, C-terminal lysine-rich region. Organ-specific expression of these genes was revealed by Northern blot analysis. AtAGP17 was strongly expressed in leaves and stems, and weakly expressed in flowers and roots; AtAGP18 was strongly expressed in flowers, and moderately expressed in roots, stems and young leaves; and AtAGP19 was strongly expressed in stems, moderately expressed in flowers and roots, and weakly expressed in young leaves. One of these genes, AtAGP17, was expressed and purified as a green fluorescent protein (GFP) fusion protein in transgenic tobacco cells using hydrophobic interaction chromatography, size exclusion chromatography and reverse phase high-performance liquid chromatography. The fusion (glyco)protein produced a characteristic AGP 'smear' with a molecular mass of 80-150 kDa when detected by Western blot analysis. Glycosyl composition and linkage analyses of purified GFP-AtAGP17 showed that carbohydrate accounted for approximately 86% of the molecule, with arabinose and galactose as major, and rhamnose and glucuronic acid as minor glycosyl residues and with 1,3,6-galactose, 1,4-glucuronic acid, 1,3-galactose and terminal arabinose as major linkages. GFP-AtAGP17 was also precipitated by beta-Yariv reagent, further confirming that AtAGP17 is a bona fide AGP. Confocal fluorescence microscopy of plasmolysed, transformed cells indicated that AtAGP17 is localized on the plasma membrane and in Hechtian strands. Hydroxyproline (Hyp) glycoside profiles of GFP-AtAGP17 in conjunction with the deduced protein sequence also served to corroborate the Hyp contiguity hypothesis, which predicts contiguous Hyp residues as attachment sites for arabinosides and clustered, non-contiguous Hyp residues as attachment sites for arabinogalactan polysaccharides.     

Cloning and sequence analysis of a cDNA encoding ferric leghemoglobin reductase from soybean nodules.

A cDNA encoding soybean (Glycine max [L.] Merr) ferric leghemoglobin reductase (FLbR), an enzyme that is postulated to play an important role in maintaining leghemoglobin in its functional ferrous state, has been cloned and characterized. A group of highly degenerate oligonucleotides deduced from the N-terminal amino acid sequence of FLbR was used to prime the polymerase chain reaction (PCR) on soybean nodule mRNA and cDNA. A full-length clone of FLbR cDNA was isolated by screening a lambda gt11 soybean nodule cDNA library using the specific PCR-amplified FLbR cDNA fragment as a probe. The cDNA contained about 1.8 kb and had a coding sequence for 523 amino acids with a predicted molecular mass of 55,729 D, which included a putative 30-residue signal peptide and a 493-residue mature protein. Computer-aided analysis of the deduced FLbR amino acid sequence showed considerable homology (varied from 20-50% with enzymes and species) to dihydrolipoamide dehydrogenase (EC 1.8.1.4), glutathione reductase (EC 1.6.4.2), mercuric reductase (EC 1.16.1.1), and trypanothione reductase (EC 1.6.4.8) in a superfamily of pyridine nucleotide-disulfide oxidoreductases from various organisms. Northern blot analysis using FLbR cDNA as a probe showed that the FLbR gene was expressed in soybean nodules, leaves, roots, and stems, with a greater level of expression in nodules and leaves than in roots and stems. Southern blot analysis of the genomic DNA showed the presence of two homologous FLbR genes in the soybean genome.     

Medicago truncatula Mt-ZFP1 encoding a root enhanced zinc finger protein is regulated by cytokinin, abscisic acid and jasmonate, but not cold.

A Medicago truncatula zinc finger protein cDNA (Mt-ZFP1) was isolated from a M.truncatula seedling cDNA library using RT-PCR product as a probe. The predicted amino acid sequence of Mt-ZFP1 is over 79% similar to S-SCOF-1 from soybean, a novel cold-inducible zinc finger protein involved in cold stress signal transduction mediated by abscisic acid (ABA). The secondary structure of Mt-ZFP1 protein was almost identical to that of S-SCOF-1. Mt-ZFP1 also contained a typical C2H2-type zinc finger domain and a putative nuclear located signal. RNA gel blot hybridization demonstrated that the Mt-ZFP1 gene was actively expressed in roots, with a lower abundance in leaf and stem tissues. Cold treatment did not induce the expression of Mt-ZFP1 in either leaves and stems or roots. Exogenous application of cytokinins marginally increased the accumulation of Mt-ZFP1 mRNA, while ABA and jasmonate treatments decreased the levels of Mt-ZFP1 mRNA. DNA gel-blot analysis demonstrated that Mt-ZFP1 is present as a single copy gene in the M. truncatula genome. These data suggest that Mt-ZFP1 is a novel zinc finger protein with different physiological functions to that of S-SCOF-1. The similar cold-inducible factor like S-SCOF-1 might not exist in M. truncatula.     

Characterization and localization of laccase forms in stem and cell cultures of sycamore

A laccase-type polyphenoloxidase (EC 1.10.3.2.), abundantly secreted by suspension-cultured sycamore (Acer pseudoplatanus) cells was purified to homogeneity. This laccase form is a glycoprotein (molecular weight 110000) with high mannose and complex glycans. The polypeptide moiety has a molecular weight of 66 000, indicating that the glycoprotein is 40% carbohydrate. Laccase is abundantly present in both the cell wall and the culture medium of suspension-cultured sycamore cells, but it is not detected in the cytoplasm, indicating that this large protein is efficiently secreted by the cells. Polyclonal rabbit antiserum was raised against the deglycosylated protein and was used to probe extracts of sycamore stem tissues. A second laccase form (molecular weight 56 000), antigenically related to laccase from cell cultures, is abundant in the epidermis of sycamore stems. In addition, this 56 kDa laccase form co-localizes with lignin precursors on tissue prints from sycamore stems. A polypeptide (molecular weight 50 000-56 000), antigenically related to sycamore laccase, was also immunodetected in most plant organs previously described in the literature as polyphenoloxidase-rich.     

Molecular characterization of tobacco mitochondrial L-galactono-gamma-lactone dehydrogenase and its expression in Escherichia coli

A cDNA clone encoding L-galactono-gamma-lactone (GAL) dehydrogenase (EC 1.3.2.3) was isolated from tobacco leaves. The cDNA clone contained an open reading frame encoding the protein of 501 amino acids with a calculated molecular mass of 56,926 Da, preceded by a putative mitochondrial targeting signal consisting of 86 amino acid residues. In fact, GAL dehydrogenase was localized in the mitochondria of tobacco cells. The deduced amino acid sequence of the cDNA showed 77 and 82% homology to cauliflower and sweet potato GAL dehydrogenases, respectively. Southern blot analysis showed that tobacco contains one copy of the gene for the enzyme. Northern blot analysis showed that GAL dehydrogenase mRNA (2.0 kb) is expressed in the leaves, stems, and roots in almost equal quantities. We introduced the cDNA clone encoding tobacco GAL dehydrogenase into a pET expression vector to overexpress this protein in Escherichia coli. The partially purified recombinant enzyme was used for comparative studies on the native enzymes from tobacco and other sources; its enzymatic properties were similar to those of other GAL dehydrogenases.     

Tissue-Specific Expression of Cell Wall Proteins in Developing Soybean Tissues

Cell wall hydroxyproline-rich glycoproteins (HRGPs) and glycine-rich proteins (GRPs) were examined at the protein and at the mRNA levels in developing soybean tissues by tissue print immunoblots and RNA blots. In young soybean stems, HRGPs are expressed most heavily in cambium cells, in a few layers of cortex cells surrounding primary phloem, and in some parenchyma cells around the primary xylem, whereas GRPs are highly expressed in the primary xylem and also in the primary phloem. In older soybean stems, HRGP genes are expressed exclusively in cambium cells and GRP genes are most heavily expressed in newly differentiated secondary xylem cells. Similar expression patterns of HRGPs and of GRPs were found in soybean petioles, seedcoats, and young hypocotyls, and also in bean petioles and stems. HRGPs and GRPs become insolubilized in soybean stem cell walls. Three major HRGP mRNAs and two major GRP mRNAs accumulate in soybean stems. Soluble HRGPs are abundant in young hypocotyl apical regions and young root apical regions, whereas in hypocotyl and root mature regions, soluble HRGPs are found only in a few layers of cortex cells surrounding the vascular bundles. GRPs are specifically localized in primary xylem cell walls of young root. These results show that the gene expression of HRGPs and GRPs is developmentally regulated in a tissue-specific manner. In soybean tissues, HRGPs are most heavily expressed in meristematic cells and in some of those cells that may be under stress, whereas GRPs are expressed in all cells that are or are going to be lignified.