Kevin S. Gould

Analysis of Nitric Oxide Signaling Functions in Tobacco Cells Challenged by the Elicitor Cryptogein

Nitric oxide (NO) has recently emerged as an important cellular mediator in plant defense responses. However, elucidation of the biochemical mechanisms by which NO participates in this signaling pathway is still in its infancy. We previously demonstrated that cryptogein, an elicitor of tobacco defense responses, triggers a NO burst within minutes in epidermal sections from tobacco leaves (Nicotiana tabacum cv Xanthi). Here, we investigate the signaling events that mediate NO production, and analyze NO signaling activities in the cryptogein transduction pathway. Using flow cytometry and spectrofluorometry, we observed that cryptogein-induced NO production in tobacco cell suspensions is sensitive to nitric oxide synthase inhibitors and may be catalyzed by variant P, a recently identified pathogen-inducible plant nitric oxide synthase. NO synthesis is tightly regulated by a signaling cascade involving Ca2+ influx and phosphorylation events. Using tobacco cells constitutively expressing the Ca2+ reporter apoaequorin in the cytosol, we have shown that NO participates in the cryptogein-mediated elevation of cytosolic free Ca2+ through the mobilization of Ca2+ from intracellular stores. The NO donor diethylamine NONOate promoted an increase in cytosolic free Ca2+ concentration, which was sensitive to intracellular Ca2+ channel inhibitors. Moreover, NO appears to be involved in the pathway(s) leading to the accumulation of transcripts encoding the heat shock protein TLHS-1, the ethylene-forming enzyme cEFE-26, and cell death. In contrast, NO does not act upstream of the elicitor-induced activation of mitogen-activated protein kinase, the opening of anion channels, nor expression of GST, LOX-1, PAL, and PR-3 genes. Collectively, our data indicate that NO is intimately involved in the signal transduction processes leading to cryptogein-induced defense responses.

A unified explanation for anthocyanins in leaves

Abstract The leaves from many of New Zealands native species are remarkably polymorphic for anthocyanin expression. Red coloration varies not only as a function of seasonal and developmental factors, but can also differ among individuals of a population, among leaves within a canopy, and even among tissues within a leaf. Moreover, the biosynthesis of anthocyanin in these leaves can be induced by a host of disparate environmental and biotic stimuli. Any unified explanation for the presence of anthocyanins in leaves must accommodate both the variability in pigmentation patterns over time and space, and the diverse range of triggers. Our data indicate that anthocyanins confer a phytoprotective role, rather than being the default end-product of a saturated flavonoid metabolism. Anthocyanins are primarily associated with chlorophyllous tissues, and significantly modify both the quantity and the quality of light incident on a chloroplast. Red leaves photosynthesise less than green leaves, but are also photoinhibited less and recover sooner following exposure to high light fluxes. Photoabatement also reduces the generation of free radicals and reactive oxygen species from photooxidation, photorespiration, and Mehler reaction activities. Anthocyanins inhibit Fenton hydroxyl radical generation by chelating to ferrous ions, and effectively scavenge superoxide and hydrogen peroxide generated by mechanical injury, sudden temperature changes, and exposures to high light. Anthocyanins are evidently versatile and highly effective phytoprotectants. However, there is probably no unified explanation for their presence in leaves. Common among the first land plants, anthocyanins have probably been hijacked over the course of evolution to perform an array of tasks.

Anthocyanins in Leaves and Other Vegetative Organs: An Introduction

Abstract Although anthocyanins are most recognized as pigments contributing to coloration in fruits and flowers, they are also present in leaves and other vegetative organs. Although their presence has long been recognized, particularly because of their contribution to autumn coloration, the phenomenon has been poorly studied and is not well understood. In this chapter we review the history of research on anthocyanins in leaves, emphasizing the flurry of research at the end of the 19 th century as well as the growing body of contemporary research on the topic. We emphasize the various hypotheses of anthocyanin function that were mainly developed more than a century ago, and emphasize recent research that takes advantage of our dramatically increased understanding of whole plant physiology.

Cytoplasmic accumulation of flavonoids in flower petals and its relevance to yellow flower colouration

It is widely accepted that the mix of flavonoids in the cell vacuole is the source of flavonoid based petal colour, and that analysis of the petal extract reveals the nature and relative levels of vacuolar flavonoid pigments. However, it has recently been established with lisianthus flowers that some petal flavonoids can be excluded from the vacuolar mix through deposition in the cell wall or through complexation with proteins inside the vacuole, and that these flavonoids are not readily extractable. The present work demonstrates that flavonoids can also be compartmented within the cell cytoplasm. Using adaxial epidermal peels from the petals of lisianthus (Eustoma grandiflorum), Lathyrus chrysanthus and Dianthus caryophyllus, light and laser scanning confocal microscopy studies revealed a significant concentration of petal flavonoids in the cell cytoplasm of some tissues. With lisianthus, flavonoid analyses of isolated protoplasts and vacuoles were used to establish that ca 14% of petal flavonoids are located in the cytoplasm (cf. 30% in the cell wall and 56% in the vacuole). The cytoplasmic flavonoids are predominantly acylated glycosides (cf. non-acylated in the cell wall). Flavonoid aggregation on a cytoplasmic protein substrate provides a rational mechanism to account for how colourless flavonoid glycosides can produce yellow colouration in petals, and perhaps also in other plant parts. High vacuolar concentrations of such flavonoids are shown to be insufficient.

Cell wall sited flavonoids in lisianthus flower petals

Flavonoids are considered to be located predominantly in the vacuoles of epidermal cells and in the cuticular wax of terrestrial plants. However, recent reports have suggested that flavonoids may also reside elsewhere in the cells of green leaves. In the present study of lisianthus flower petals, it is demonstrated that ca. 30% of the whole petal flavonol glycosides are located in the cell wall. These flavonol glycosides are distinguished from the vacuolar glycosides in that they lack acylation. Evidence from light and confocal microscopy studies is corroborated by HPLC analyses of isolated protoplasts and cell wall digests, these having been produced by enzymic treatment of epidermal peels. This is the first report of the occurrence of flavonoids in petal cell walls, and it describes novel methodology for such studies.

Do anthocyanins protect leaves of New Zealand native species from UV-B?

Abstract Anthocyanin pigments must reside in the uppermost tissues of a leaf if they are to be effective as UV -B filters. However, in our survey of leaves and phylloclades from 25 native New Zealand plants, only four species held anthocyanins in the upper epidermis and/or hypodermis. For 18 species, anthocyanins were located in vacuoles of the palisade and/or spongy mesophyll, the same tissues that are potentially susceptible to UV-B-induced photoinhibition. Leaf pigmentation patterns varied among species and were correlated to the histological distributions ofanthocyanins. Most species held cyanidin-derived pigments. UV-B filtration cannot be regarded as a unified theory for anthocyanin function in leaves.

NO signaling functions in the biotic and abiotic stress responses

Over the past two decades, it has been recognized that nitric oxide (NO) plays an important role in diverse mammalian physiological processes. NO regulates physiological processes by modulating the activity of proteins principally by nitrosylation, a process referring to the binding of NO to a transition metal centre or cysteine residues [1]. An important class of proteins that constitutes key targets of NO is that of the Ca2+ channels including plasma membranes as well intracellular Ca2+ channels. NO modulates these channels directly by nitrosylation, but also indirectly via the second messenger cyclic GMP (cGMP) and/or cyclic ADP ribose (cADPR). Therefore, NO emerges as a key messenger governing the overall control of Ca2+ homeostasis [2]. In the late 1990s, NO also became an increasingly popular target for investigation in plants. As in mammals, NO fulfils a broad spectrum of signaling functions in (patho)physiological processes in plants [3]. Here, we summarise studies published in recent years that provide novel insights into the signaling functions of NO produced by plant cells exposed to abiotic stresses and biotic stress (pathogen-derived elicitors). It focuses particularly on the cross-talk operating between NO and Ca2+.

Leaf orientation and light interception by juvenile Pseudopanax crassifolius (Cunn.) C. Koch in a partially shaded forest environment

Leaf orientations and light environments were recorded for 40 juvenile Pseudopanax crassifolius trees growing in New Zealand in a partially shaded, secondary forest environment. Efficiencies of interception of diffuse and direct light by the observed leaf arrangments were calculated relative to those of three hypothetical leaf arrangements. Canopy gaps above the study plants were unevenly distributed with respect to azimuth and elevation above the horizon. Our results indicate that photosynthetically active radiation (PAR) received from the sides is more important than that received from directly above. In 33 of the plants leaf orientation was found to be significantly clustered towards one azimuth. The mean azimuth and the mean angle of declination were different for each plant. Leaves were steeply declined, and oriented towards the largest canopy gap at each site. Steep leaf angles reduced interception of direct and diffuse PAR when compared to interception by plant with a hypothetical horizontal leaf arrangement. When compared to a hypothetical arrangement with steep leaf declination and a uniform azimuth distribution, the observed leaf arrangement increased the efficiency of interception of diffuse PAR, but had a variable effect on the interception of direct PAR. Results indicate that the developing leaves of juvenile P. crassifolius orient towards the strongest sources of diffuse light, regardless of their value as a source of direct light. By maximising diffuse light interception while reducing direct light interception, leaf orientation may be a partial determinant of the types of habitats exploited by this species. This study emphasises the importance of considering diffuse light interception for plants growing in partially shaded environments.

Floral biology and breeding system of pohutukawa (Metrosideros excelsa, Myrtaceae)

Abstract The floral biology and breeding system of pohutukawa (Metrosideros excelsa, Myrtaceae), a mass‐flowering tree of northern New Zealand coastlines, were examined. Trees flower over a peak period of 2 weeks, and compound inflorescences contain an average of 14.3 showy, hermaphrodite, red brush flowers that remain open for 7 days. A brief female flower stage (mean duration 1.3 d) is followed by the main hermaphrodite phase that lasts for 4 days. Neither dichogamy nor herkogamy is important in preventing pollen and stigma interference. Pollen is highly viable (93.6%), and stigma receptivity extends for at least 9 days, as indicated by peroxidase activity, pollen germination, pollen tube length 24 h after pollination, and seed set. Stigmatic exudate production appears to increase up to 5 days post‐anthesis. On average, flowers produce 46 p1 nectar per day, containing 18% (w/v) sucrose. Floral design and display of pohutukawa are consistent with high levels of autogamous and geitonogamous self‐pollinati...

Rootstock effects on budburst and flowering in kiwifruit

Effects of five Actinidia rootstocks on budburst and flowering characteristics of ‘Hayward’ kiwifruit scions were examined. Compared with a standard clonal Actinidia deliciosa rootstock, clones of Actinidia hemsleyana, Actinidia eriantha and Actinidia rufa increased ‘Hayward’ flower numbers per cane by 110%, 73% and 30%, respectively. This occurred principally as a result of increases in the mean number of flowers per shoot. The Actinidia chinensis rootstock however, reduced flowering by 23% compared with the standard rootstock. The numbers of floral primordia initiated in buds were similar in the three ‘Hayward’/rootstock combinations which were examined in more detail. Rootstock effects on flower number at anthesis were therefore, owing to effects on the level of floral abortion after flower initiation. Rootstocks also affected budburst patterns of ‘Hayward’ scions. Budburst was highest and most synchronous on ‘Hayward’/ A. hemsleyana vines, while ‘Hayward’/ A. chinensis vines had lowest and least synchronous budburst. Both the magnitude and synchrony of budburst were highly correlated with flower number per shoot associated with a particular rootstock.