Arabinogalactan protein and wall-associated kinase in a plasmalemmal reticulum with specialized vertices

Summary Arabinogalactan protein and wall-associated kinase (WAK) are suspected to be regulatory players at the interface between cytoplasm and cell wall. Both WAK(s) and arabinogalactan shown likely to represent arabinogalactan protein(s) have been visualized there with computational optical-sectioning microscopy. The arabinogalactan occurs in a polyhedral array at the external face of the cell membrane. WAK, and other proteins as yet unidentified, appear to fasten the membrane to the wall at vertices of the array. Evidence is presented that the array bears an important part of the mechanical stress experienced by the membrane, and it is speculated that the architectural organization of arabinogalactan protein, WAK, and other components of the array is critical for coordination of endomembrane activities, growth, and differentiation. The array has been named the plasmalemmal reticulum.Abbreviations AGP arabinogalactan protein - (-D-Glc)₃ -D-glucosyl Yariv phenylglycoside - (-D-Man)₃ -D-mannosyl Yariv phenylglycoside - SDS-AGE sodium dodecyl sulfate-agarose gel electrophoresis - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - WAK wall-associated kinase     

Antisense suppression of violaxanthin de-epoxidase in tobacco does not affect plant performance in controlled growth conditions

Violaxanthin de-epoxidase (VDE) catalyzes the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin in the xanthophyll cycle. Tobacco was transformed with an antisense VDE construct under control of the cauliflower mosaic virus 35S promoter to determine the effect of reduced levels of VDE on plant growth. Screening of 40 independent transformants revealed 18 antisense lines with reduced levels of VDE activity with two in particular (TAS32 and TAS39) having greater than 95% reduction in VDE activity. Northern analysis demonstrated that these transformants had greatly suppressed levels of VDE mRNA. De-epoxidation of violaxanthin was inhibited to such an extent that no zeaxanthin and only very low levels of antheraxanthin could be detected after exposure of leaves to high light (2000 μmol m⁻² s⁻¹ for 20 min) with no observable effect on levels of other carotenoids and chlorophyll. Non-photochemical quenching was greatly reduced in the antisense VDE tobacco, demonstrating that a significant level of the non-photochemical quenching in tobacco requires de-epoxidation of violaxanthin. Although the antisense plants demonstrated a greatly impaired de-epoxidation of violaxanthin, no effect on plant growth or photosynthetic rate was found when plants were grown at a photon flux density of 500 or 1000 μmol m⁻² s⁻¹ under controlled growth conditions as compared to wild-type tobacco. This revised version was published online in June 2006 with corrections to the Cover Date.     

Splicing of Arabidopsis tRNAMet precursors in tobacco cell and wheat germ extracts

Intron-containing tRNA genes are exceptional within nuclear plant genomes. It appears that merely two tRNA gene families coding for tRNA^Tyr _{G A} and elongator tRNA^Met _CmAU contain intervening sequences. We have previously investigated the features required by wheat germ splicing endonuclease for efficient and accurate intron excision from Arabidopsis pre-tRNA^Tyr. Here we have studied the expression of an Arabidopsis elongator tRNA^Met gene in two plant extracts of different origin. This gene was first transcribed either in HeLa or in tobacco cell nuclear extract and splicing of intron-containing tRNA^Met precursors was then examined in wheat germ S23 extract and in the tobacco system. The results show that conversion of pre-tRNA^Met to mature tRNA proceeds very efficiently in both plant extracts. In order to elucidate the potential role of specific nucleotides at the 3 and 5 splice sites and of a structured intron for pre-tRNA^Met splicing in either extract, we have performed a systematic survey by mutational analyses. The results show that cytidine residues at intron-exon boundaries impair pre-tRNA^Met splicing and that a highly structured intron is indispensable for pre-tRNA^Met splicing. tRNA precursors with an extended anticodon stem of three to four base pairs are readily accepted as substrates by wheat and tobacco splicing endonuclease, whereas pre-tRNA molecules that can form an extended anticodon stem of only two putative base pairs are not spliced at all. An amber suppressor, generated from the intron-containing elongator tRNA^Met gene, is efficiently processed and spliced in both plant extracts.     

Heterotrimeric G-protein β-subunit is localized in the plasma membrane and nuclei of tobacco leaves

Heterotrimeric G-proteins are involved in a variety of cellular responses, but relatively little is known about their function and biochemistry in plants. Antibodies raised against the tobacco heterotrimeric G-protein -subunit (G) were used to analyse its distribution in tobacco leaves. In young tissue the protein level was relatively high, while it declined substantially during later stages of leaf development. Cell fractionation revealed that G is tightly associated with plasma membrane, but can also be detected in purified nuclei.     

Isolation and characterization of two pathogen- and salicylic acid-induced genes encoding WRKY DNA-binding proteins from tobacco

A pathogen- and salicylic acid (SA)-induced DNA-binding activity has been recently identified in tobacco that is related to a previously identified class of WRKY DNA-binding proteins. To identify members of the WRKY gene family associated with this DNA-binding activity, we have attempted to isolate those WRKY genes that are induced by pathogen infection. Using a domain-specific differential display procedure, we have isolated two tobacco WRKY genes, tWRKY3 and tWRKY4, that are rapidly induced in resistant tobacco plants after infection by tobacco mosaic virus (TMV). Both tWRK3 and tWRKY4 encode proteins with a single WRKY domain that contain the conserved WRKYGQK sequence. Unlike other isolated WRKY proteins that contain the Cys₂His₂ zinc motif, tWRKY3 and tWRKY4 appear to contain the Cys₂HisCys zinc motif. Nonetheless, both tWRKY3 and tWRKY4 are capable of binding DNA molecules with the W-box (TTGAC) element recognized by other WRKY proteins. Expression of the tWRKY3 and tWRKY4 genes could be rapidly induced not only by TMV infection but also by SA or its biologically active analogues that are capable of inducing pathogenesis-related genes and enhanced resistance. Interestingly, induction of both genes by TMV infection was still observed in resistant tobacco plants expressing the bacterial salicylate hydroxylase gene (nahG), although the levels of induction appeared to be reduced. Identification of pathogen- and SA-induced genes encoding WRKY DNA-binding proteins should facilitate future studies on the regulation and functions of this novel group of DNA-binding proteins.     

Cloning and sequence analysis of lily and tobacco guanylate kinases

Guanylate kinase is an essential enzyme in the nucleotide biosynthetic pathway, catalyzing the reversible transfer of the terminal phospharyl group of ATP to GMP or dGMP. This enzyme has been well studied from several organisms and many structural and functional details have been characterized. Animal GMP kinases have also been implicated in signal transduction pathways. However, the corresponding role by plant derived GMP kinases remains to be elucidated. Full-length cDNA clones encoding enzymatically active guanylate kinases were isolated from cDNA libraries of lily and tobacco. Lily cDNA is predicted to encode a 392-amino acid protein with a molecular mass of 43.1 kDa and carries amino- and carboxy- terminal extensions of the guanylate kinase (GK)-like domain. But tobacco cDNA is predicted to encode a smaller protein of 297-amino acids with a molecular mass of 32.7 kDa. The amino acid residues known to participate in the catalytic activity of functionally characterized GMP kinases, are also conserved in GK domains of LGK-1 and NGK-1. The GK domains of NGK-1, LGK-1 and previously characterized AGK-1 from Arabidopsis exhibit 74–84% identity, whereas their N- and C-terminal domains are more divergent with amino acid conservation in the order of 48-55%. Phylogenetic analysis on the deduced amino acid sequences reveals that NGK-1 and LGK-1 form one distinct subgroup along with AGK-1 and AGK-2 homologues from Arabidopsis. Isolation of GMP kinases from diverse plant species like lily and tobacco adds a new dimension in understanding their role in cell signaling pathways that are associated with plant growth and development.     

Transient expression and heat-stress-induced co-aggregation of endogenous and heterologous small heat-stress proteins in tobacco protoplasts

Heat-stress granules (HSG) are highly ordered, cytoplasmic chaperone complexes found in all heat-stressed plant cells. We have developed an experimental system involving expression of cytosolic class I and class II small heat-stress proteins (Hsps) of pea, Arabidopsis and tomato in tobacco protoplasts to study the structural prerequisites for the assembly of HSG or HSG-like complexes. Class I and class II small Hsps formed class-specific dodecamers of 210-280 kDa, which, upon heat stress, were incorporated into HSG complexes. Interestingly, class II dodecamers alone could form HSG-like complexes (auto-aggregation), whereas class I dodecamers could do so only in the presence of class II proteins (recruitment). By analysing C-terminal deletion forms of Hsp17 class II, we obtained evidence that the intact C-terminus is critical for the oligomerization state, for the heat-stress-induced auto-aggregation and for recruitment of class I proteins. The class-specific formation of dimers as a prerequisite for oligomerization was analysed by the yeast two-hybrid system. In the presence of the endogenous (tobacco) set of heat-stress-induced proteins, all heterologous class I and class II proteins were incorporated into HSG complexes, whose ultrastructure was different from that of complexes formed by class I and class II proteins alone. Although other, more distantly related, members of the Hsp20 family, i.e. the plastidic pea Hsp21, the Drosophila Hsp23 and the mouse Hsp25, were well expressed in tobacco protoplasts and formed homo-oligomers of 200-700 kDa, none of them could be recruited to HSG complexes.     

Adaptation of Spodoptera exigua (Lepidoptera: Noctuidae) to barley trypsin inhibitor BTI-CMe expressed in transgenic tobacco

Nicotiana tabacum plants were transformed with the cDNA of barley trypsin inhibitor BTI-CMe under the control of the 35S CaMV promoter. Although the transgene was expressed and the protein was active in the homozygous lines selected, growth of Spodoptera exigua (Lepidoptera: Noctuidae) larvae reared on transgenic plants was not affected. The protease activity in larval midgut extracts after 2 days feeding on transformed tobacco leaves from the highest expressing plant showed a reduction of 25% in the trypsin-like activity compared to that from insects fed on non-transformed controls. The susceptibility of digestive serine-proteases to inhibition by BTI-CMe was confirmed by activity staining gels. This decrease was compensated with a significant induction of leucine aminopeptidase-like and carboxipeptidase A-like activities, whilechymotrypsin-, elastase-, and carboxipeptidase B-like proteases were not affected.     

Distribution and residual activity of two insecticidal proteins, avidin and aprotinin, expressed in transgenic tobacco plants, in the bodies and frass of Spodoptera litura larvae following feeding

To understand how a major cosmopolitan pest responds to two very different insecticidal proteins and to determine whether herbivorous insects and their frass could be environmental sources of recombinant proteins from transgenic plants, Spodoptera litura (Fab.) (Lepidoptera, Noctuidae) larvae were fed on tobacco leaves expressing either the biotin-binding protein, avidin, or the protease inhibitor, aprotinin. Control larvae received non-transgenic tobacco. Samples of larvae were taken after 5, 6 or 7 days’ feeding and frass was collected after two 24-h periods at 6 and 7 days. Insects in all treatments grew significantly during the experiment, but the avidin-fed larvae were significantly smaller than the others on Day 7. Avidin was found in all samples of avidin-fed larvae (7.0±0.86 ng mg⁻¹, n=45), at a lower level than in their frass (31.9±5.08 ng mg⁻¹, n=30), and these frass levels were lower than those of the the leaves fed to the larvae (69.0±6.71 ng mg⁻¹, n=45). All of the avidin detected in these samples was capable of binding biotin. On average, between 10 and 28% of avidin was recovered with the methods used, whereas almost full recovery of aprotinin was effected. Aprotinin levels in larvae (8.2±0.53 ng mg⁻¹, n=45) were also lower than aprotinin levels in frass (77.4±6.9 ng mg⁻¹, n=30), which were somewhat lower than those in the leaves fed to the larvae (88.6±2.51 ng mg⁻¹, n=45). Approximately half the trypsin-binding ability of aprotinin was lost in larvae, and in frass, aprotinin had lost about 90% of its ability to bind trypsin.     

Nitrogen supply after removing the shoot apex increases the nicotine concentration and nitrogen content of tobacco plants

BACKGROUND: and Aims High nicotine concentrations in leaves, especially in the upper leaves, offer a serious problem for the cultivation of tobacco (Nicotiana tabacum). Preliminary field experiments showed that rapid mineralization of soil N during late stages of growth may contribute to high nicotine concentrations in leaves. METHODS: A sand-culture experiment was carried out in the greenhouse. The N supply was controlled during the experiment, and different amounts of 15N were supplied during late stages of growth (after removal of the shoot apex), to investigate the contribution of the N taken up at this time to the N content of and nicotine concentration in tobacco plants. KEY RESULTS: Addition of 1.6 g or 4 g 15N-labelled NH4NO3 after removing the shoot apex and flushing out the 14N did not increase leaf dry weights; however, it did result in delayed leaf senescence, more lateral bud formation, and an increase in 15N as a proportion of total N, and nicotine-15N as a proportion of total nicotine-N in each organ. The nicotine concentration, 15N and nicotine-15N abundances were increased from the bottom to the top leaves. When more 15N-labelled NH4NO3 was supplied, the nicotine concentration in leaves increased, and so did the 15N abundance in nicotine-N. CONCLUSION: Enhanced N supply in the later growth stages (after removing the apex) increased N content and nicotine concentration in tobacco plants. Nicotine was synthesized de novo during the late growth stages.