A major function of the tobacco floral nectary is defense against microbial attack

 We have characterized the major nectar protein (Nectarin I) from ornamental tobacco as a superoxide dismutase that functions to generate high levels of hydrogen peroxide in nectar. Other nectar functions include an anti-polygalacturonase activity that may be due to a polygalacturonase inhibiting protein (PGIP). We also examined the expression of defense related genes in the nectary gland by two independent methods. We isolated a sample of nectary-expressed cDNAs and found that 21% of these cDNAs were defense related clones. Finally, we examined the expression of a number of specific defense-related genes by hybridization to specific cDNAs. These results demonstrated that a number of specific defense genes were more strongly expressed in the floral nectary than in the foliage. Taken together these results indicate that the floral nectary gland can have specific functions in plant defense. Received August 8, 2002; accepted January 7, 2003 Published online: June 2, 2003     

Tobacco nectaries express a novel NADPH oxidase implicated in the defense of floral reproductive tissues against microorganisms

Hydrogen peroxide produced from the nectar redox cycle was shown to be a major factor contributing to inhibition of most microbial growth in floral nectar; however, this obstacle can be overcome by the floral pathogen Erwinia amylovora. To identify the source of superoxide that leads to hydrogen peroxide accumulation in nectary tissues, nectaries were stained with nitroblue tetrazolium. Superoxide production was localized near nectary pores and inhibited by diphenylene iodonium but not by cyanide or azide, suggesting that NAD(P)H oxidase is the source of superoxide. Native PAGE assays demonstrated that NADPH (not NADH) was capable of driving the production of superoxide, diphenyleneiodonium chloride was an efficient inhibitor of this activity, but cyanide and azide did not inhibit. These results confirm that the production of superoxide was due to an NADPH oxidase. The nectary enzyme complex was distinct by migration on gels from the leaf enzyme complex. Temporal expression patterns demonstrated that the superoxide production (NADPH oxidase activity) was coordinated with nectar secretion, the expression of Nectarin I (a superoxide dismutase in nectar), and the expression of NOX1, a putative gene for a nectary NADPH oxidase that was cloned from nectaries and identified as an rbohD-like NADPH oxidase. Further, in situ hybridization studies indicated that the NADPH oxidase was expressed in the early stages of flower development although superoxide was generated at later stages (after Stage 10), implicating posttranslational regulation of the NADPH oxidase in the nectary.     

Identification, cloning and characterization of a GDSL lipase secreted into the nectar of Jacaranda mimosifolia

The presence and function of several proteins secreted into floral nectars has been described in recent years. Here we report the presence of at least eight distinct proteins secreted into the floral nectar of the tropical tree Jacaranda mimosifolia (Bignoniaceae). Steps were initiated to identify and characterize these proteins in order to determine potential functions. The N-terminal sequence of the major Jacaranda nectar protein, JNP1, at 43 kDa contained similarity with members of the plant GDSL lipase/esterase gene family. Based upon this sequence, a full-length cDNA was isolated and predicted to encode a mature protein of 339 amino acids with a molecular mass of 37 kDa. Both raw nectar and heterologously expressed JNP1 displayed lipase/esterase activities. Interestingly, J. mimosifolia flowers produce an opaque, white colored nectar containing spherical, lipophilic particles approximately 5 microm in diameter and smaller. GS-MS analysis also identified the accumulation of free fatty acids within the nectar. It is proposed that JNP1 hydrolyzes Jacaranda nectar lipids with the concomitant release of free fatty acids. Potential functions of JNP1 in relation to pollinator attraction and prevention of microbial growth within nectar are briefly discussed.     

Interaction of Nectarin 4 with a fungal protein triggers a microbial surveillance and defense mechanism in nectar

Understanding the biochemical mechanisms by which plants respond to microbial infection is a fundamental goal of plant science. Extracellular dermal glycoproteins (EDGPs) are widely expressed in plant tissues and have been implicated in plant defense responses. Although EDGPs are known to interact with fungal proteins, the downstream effects of these interactions are poorly understood. To gain insight into these phenomena, we used tobacco floral nectar as a model system to identify a mechanism by which the EDGP known as Nectarin IV (NEC4) functions as pathogen surveillance molecule. Our data demonstrates that the interaction of NEC4 with a fungal endoglucanase (XEG) promotes the catalytic activity of Nectarin V (NEC5), which catalyzes the conversion of glucose and molecular oxygen to gluconic acid and H(2)O(2). Significantly enhanced NEC5 activity was observed when XEG was added to nectar or nectarin solutions that contain NEC4. This response was also observed when the purified NEC4:XEG complex was added to NEC4-depleted nectarin solutions, which did not respond to XEG alone. These results indicate that formation of the NEC4:XEG complex is a key step leading to induction of NEC5 activity in floral nectar, resulting in an increase in concentrations of reactive oxygen species (ROS), which are known to inhibit microbial growth directly and activate signal transduction pathways that induce innate immunity responses in the plant.     

Floral nectary,nectar production dynamics,and floral reproductive isolation among closely related species of Pedicularis

Floral nectar is thought to be one of the most important rewards that attract pollinators in Pedicularis;however,few studies have examined variation of nectary structure and/or nectar secretion in the genus,particularly among closely related species. Here we investigated nectary morphology,nectar quality,and nectar production dynamics in flowers of Pedicularis section Cyathophora. We found a conical floral nectary at the base of the ovary in species of the rex-thamnophila clade. Stomata were found on the surface of the nectary,and copious starch grains were detected in the nectary tissues. In contrast,a semi-annular nectary was found in flowers of the species of the superba clade. Only a few starch grains were observed in tissues of the semi-annular nectary,and the nectar sugar concentration in these flowers was much lower than that in the flowers of the rexthamnophila clade. Our results indicate that the floral nectary has experienced considerable morphological,structural,and functional differentiation among closely related species of Pedicularis. This could have affected nectar production,leading to a shift of the pollination mode. Our results also imply that variation of the nectary morphology and nectar production may have played an important role in the speciation of sect. Cyathophora.     

Identification of S-RNase and peroxidase in petunia nectar

Previous SDS PAGE gel analysis of the floral nectars from petunia and tobacco plants revealed significant differences in the protein patterns. Petunia floral nectar was shown to contain a number of RNase activities by in gel RNase activity assay. To identify these proteins in more detail, the bands with RNase activity were excised from gel and subjected to trypsin digestion followed by LC-MS/MS analysis. This analysis revealed that S-RNases accumulate in nectar from Petunia hybrida, where they should carry out a biological function different from self-pollen rejection. In addition, other proteins were identified by the LC-MS/MS analysis. These proteins include a peroxidase, an endochitinase, and a putative fructokinase. Each of these proteins contained a secretory signal sequence that marked them as potential nectar proteins. We developed RT-PCR assays for each of these five proteins and demonstrated that each of these proteins was expressed in the petunia floral nectary. A discussion of the role of these proteins in antimicrobial activity in nectar is presented.     

Is nectar reabsorption restricted by the stalk cells of floral and extrafloral nectary trichomes?

Reabsorption is a phase of nectar dynamics that occurs concurrently with secretion; it has been described in floral nectaries that exude nectar through stomata or unicellular trichomes, but has not yet been recorded in extrafloral glands. Apparently, nectar reabsorption does not occur in multicellular secretory trichomes (MST) due to the presence of lipophilic impregnations – which resemble Casparian strips – in the anticlinal walls of the stalk cells. It has been assumed that these impregnations restrict solute movement within MST to occur unidirectionally and exclusively by the symplast, thereby preventing nectar reflux toward the underlying nectary tissues. We hypothesised that reabsorption is absent in nectaries possessing MST. The fluorochrome lucifer yellow (LYCH) was applied to standing nectar of two floral and extrafloral glands of distantly related species, and then emission spectra from nectary sections were systematically analysed using confocal microscopy. Passive uptake of LYCH via the stalk cells to the nectary tissues occurred in all MST examined. Moreover, we present evidence of nectar reabsorption in extrafloral nectaries, demonstrating that LYCH passed the stalk cells of MST, although it did not reach the deepest nectary tissues. Identical (control) experiments performed with neutral red (NR) demonstrated no uptake of this stain by actively secreting MST, whereas diffusion of NR did occur in plasmolysed MST of floral nectaries at the post‐secretory phase, indicating that nectar reabsorption by MST is governed by stalk cell physiology. Interestingly, non‐secretory trichomes failed to reabsorb nectar. The role of various nectary components is discussed in relation to the control of nectar reabsorption by secretory trichomes.     

荆条花蜜腺发育解剖学研究

荆条（Ｖｉｔｅｘ ｃｈｉｎｅｎｓｉｓ Ｍｉｌｌ．）花蜜腺属于淀粉型子房蜜腺,呈圆筒状环绕于子房的基部。蜜腺外观上无特殊结构,表面有。由分泌表皮和泌蜜组织组成,包括分泌表皮、气孔器、泌蜜薄壁组织和维管束。密腺和子房壁起源相同。花蕾膨大期,泌蜜组织细胞中产生大液泡;露冠期,泌蜜组织中形成维管束;花蕾初放期,分泌表皮细胞分化形成气孔器,无气孔下室,淀粉粒的积累在此期达到高峰;盛花期,蜜腺中已无淀粉粒,密     

Nectarin IV, a potent endoglucanase inhibitor secreted into the nectar of ornamental tobacco plants. Isolation, cloning, and characterization

We have isolated and characterized the Nectarin IV (NEC4) protein that accumulates in the nectar of ornamental tobacco plants (Nicotiana langsdorffii x Nicotiana sanderae var LxS8). This 60-kD protein has a blocked N terminus. Three tryptic peptides of the protein were isolated and sequenced using tandem mass spectroscopy. These unique peptides were found to be similar to the xyloglucan-specific fungal endoglucanase inhibitor protein (XEGIP) precursor in tomato (Lycopersicon esculentum) and its homolog in potato (Solanum tuberosum). A pair of oligonucleotide primers was designed based on the potato and tomato sequences that were used to clone a 1,018-bp internal piece of nec4 cDNA from a stage 6 nectary cDNA library. The remaining portions of the cDNA were subsequently captured by 5' and 3' rapid amplification of cDNA ends. Complete sequencing of the nec4 cDNA demonstrated that it belonged to a large family of homologous proteins from a wide variety of angiosperms. Related proteins include foliage proteins and seed storage proteins. Based upon conserved identity with the wheat (Triticum aestivum) xylanase inhibitor TAXI-1, we were able to develop a protein model that showed that NEC4 contains additional amino acid loops that are not found in TAXI-1 and that glycosylation sites are surface exposed. Both these loops and sites of glycosylation are on the opposite face of the NEC4 molecule from the site that interacts with fungal hemicellulases, as indicated by homology to TAXI-I. NEC4 also contains a region homologous to the TAXI-1 knottin domain; however, a deletion in this domain restructures the disulfide bridges of this domain, resulting in a pseudoknottin domain. Inhibition assays were performed to determine whether purified NEC4 was able to inhibit fungal endoglucanases and xylanases. These studies showed that NEC4 was a very effective inhibitor of a family GH12 xyloglucan-specific endoglucanase with a K(i) of 0.35 nm. However, no inhibitory activity was observed against other family GH10 or GH11 xylanases. The patterns of expression of the NEC4 protein indicate that, while expressed in nectar at anthesis, it is most strongly expressed in the nectary gland after fertilization, indicating that inhibition of fungal cell wall-degrading enzymes may be more important after fertilization than before.     

Cloning and expression of two genes encoding auxin-binding proteins from tobacco

Two genes encoding the auxin-binding protein (ABP1) of tobacco (Nicotiana tabacum L.), both of which possess the characteristics of a luminal protein of the endoplasmic reticulum (ER), were isolated and sequenced. These genes were composed of at least five exons and four introns. The two coding exons showed 95% sequence homology and coded for two precursor proteins of 187 amino acid residues with molecular masses of 21 256 and 21 453 Da. The deduced amino acid sequences were 93% identical and both possessed an amino-terminal signal peptide, a hydrophilic mature protein region with two potential N-glycosylation sites and a carboxyl-terminal sorting signal, KDEL, for the ER. Restriction mapping of the cDNAs encoding tobacco ABP1, previously purified by amplification of tobacco cDNA libraries by polymerase chain reaction (PCR) using specific primers common to both genes, indicated that both genes were expressed, although one was expressed at a higher level than the other. Genomic Southern blot hybridization showed no other homologous genes except for these two in the tobacco genome. The apparent molecular mass of the mature form of tobacco ABP1 was revealed to be 25 kDa by SDS polyacrylamide gel electrophoresis using affinity-purified anti (tobacco ABP1) antibodies raised against a fusion protein with maltose-binding protein. Expression of the recombinant ABP1 gene in transgenic tobacco resulted in accumulation of the 25 kDa protein. A single point mutation of an amino acid residue at either of the two potential N-glycosylation sites resulted in a decrease in the apparent molecular mass and produced a 22 kDa protein. Mutations at both sites resulted in the formation of a 19.3 kDa protein, suggesting that tobacco ABP1 is glycosylated at two asparagine residues.     

地椒花蜜腺发育的解剖学观察

通过显微和亚显微观察对地椒花蜜腺的发育进行了研究。地椒花蜜腺位于子房基部的花盘上,属于盘状蜜腺,新鲜时呈绿色。蜜腺由分泌表皮和泌蜜组织组成,分泌表皮为一层细胞,表皮细胞角质膜较厚,表皮上分布着大量的气孔器,气孔器突出于表面;泌蜜组织细胞多层。花盘中央有维管束通向子房,在维管束和泌蜜组织之间有４￣５层大型的薄壁细胞。蜜腺由花盘的表皮及其内侧相邻的细胞发育而来,在发育过程中,其细胞中的液泡和贮藏的淀粉     

油菜花蜜腺的发育过程及组织化学变化

油菜花蜜腺４枚,２故侧蜜腺和２枚中蜜腺。蜜腺由分泌表皮、产蜜组织和维管束组成。蜜腺原基是在花的各部分原基发生后,由花托局部区域的表面数层细胞经反分化形成。但它在短期内,完成了形态建成,与花的成熟同步。在蜜腺的发育过程中,产蜜组织细胞的液泡发生有规律的变化。同时,淀粉粒、蛋白质的动态也较为明显,以上变化与蜜汁的合成和分泌密切相关。其蜜腺的原蜜汁是由韧皮部提供,运转至产蜜组织内加工,最后由变态气孔和泌蜜通道泌出。     

沙枣花蜜腺的发育解剖学研究

沙枣的花蜜腺位于花柱基部的筒状花盘上,属花盘蜜腺,其蜜腺位于花盘外方,由分履表皮和产蜜组织组成。分泌表皮具有角质层和变态的气孔器。产蜜组织在发育过程中,其液泡和淀粉粒都随着蜜腺的发育呈现一定的消长规律,最后形成的蜜汁由盘状蜜腺表面的气孔泌出。     

Nectaries and male-biased nectar production in protandrous flowers of a perennial umbellifer <Emphasis Type="Italic">Angelica sylvestris</Emphasis> L. (Apiaceae)

Nectar is the most common floral pollinator reward. In dichogamous species, floral nectar production rates can differ between sexual phases. We studied the structure of nectaries located on the stylopodium and nectar production in protandrous umbellifer Angelica sylvestris. Our study species produced nectar in both floral sexual phases. Nectar sugar concentration was low (on average 22 ± 11 %, mean ± SD) and the nectar hexose rich and composed of sucrose, glucose, fructose and a small amount of amino acids, including β-alanine, a non-protein amino acid. Although nectar composition and sugar concentration varied little between floral sexual phases, nectar production showed a threefold reduction during the stigma receptive period. This is in contrast to other studies of Apiaceae that have reported female-biased nectar production, but in the direction predicted by plant sexual selection theory, suggesting that in pollen-unlimited species, floral rewards mainly enhance male reproductive success. The structure of the nectary was similar at the two sexual stages investigated, and composed of a secretory epidermis and several layers of nectariferous and subsecretory parenchyma. The nectary cells were small, had large nuclei, numerous small vacuoles and dense, intensely staining cytoplasm with abundant endoplasmic reticulum, mitochondria and secretory vesicles. They contained abundant resin-like material that may potentially act as defence against microbes. Starch was rarely observed in the nectary cells, occurring predominantly at the female stage and mainly in guard and parenchyma cells in close proximity to stomata, and in subsecretory parenchyma. The main route of nectar release in A. sylvestris seems to be via modified stomata.