Introduction

Most reports on effects of enhanced UV-B radiation on terrestrial plants relate to agricultural crops cultivated under laboratory, climate room or greenhouse conditions (Caldwell & Flint, 1994a). Many early studies reported differential growth reduction and a decrease in yield of crops, mostly from temperate climate regions. In reviews by Caldwell, Terramura & Tevini (1989) and Caldwell and Flint (1994b), direct damage effects were stressed. Reduced plant growth under enhanced UV-B was expressed as a reduction of plant height, plant dry weight and leaf area. Also, photosynthetic activity was reported to be reduced under enhanced UV-B radiation, through direct effects on the photosynthetic process (photosystem II, in particular) or indirectly by effects of UV-B radiation on photosynthetic pigments or stomatal functioning. Sensitivity to enhanced UV-B was shown to vary among plant species and cultivars of a crop species.

Fewer studies have been made of UV-B effects on native plant species in their natural ecosystems. Where these have been carried out, negative effects of enhanced UV-B radiation, simulating realistic scenarios of stratospheric ozone depletion, tend to be less than predicted from greenhouse studies (see chapter by Corlett et al., this volume). Rather than direct effects of enhanced solar UV-B radiation, indirect UV-B effects may change structure and functioning of agro-ecosystems and natural ecosystems. This chapter considers the effects of enhanced UV-B radiation on plants from agro-ecosystems and natural ecosystems.

Introduction

The effects of UV-B on interactions between plants and their consumers, herbivores and the micro-organisms that cause disease or bring about decomposition of dead tissues, have been the subject of speculation for a number of years (Caldwell et al, 1989), and are increasingly seen to be of potential importance in assessing the consequences of ozone depletion on agriculture and natural ecosystems. Unfortunately, the possible importance of host–consumer interactions in determining the impacts of ozone depletion remain very difficult to assess owing to the scarcity of relevant experimental investigations. It has been hypothesised that increasing UV-B will alter plant–consumer interactions owing to changes in secondary plant metabolism (for example, Caldwell et al., 1995). While this hypothesis points to a rather consistent decrease in herbivory, disease or decomposition, no such consistency is apparent in the few studies which have been published. Indeed, current data tends to highlight the great diversity in responses to UV-B. In this chapter the diversity of responses which have been observed, and the mechanisms which might underlie these variations, will be considered, and will concentrate here on the effects of UV-B on fungi which occur as saprotrophs in the phylloplane and, especially, which cause plant disease, since a) there is slightly more published literature relating to the UV-B responses of these organisms, and b) they have been the main focus of studies at Lancaster.

Introduction

Ozone-depleting substances as well as global climatic changes (for example, increasing ‘greenhouse’ gases) may alter ozone chemistry by creating conditions that facilitate an increased ozone degradation. It is also of concern that ozone reductions are not confined to the Antarctic, but extend to mid-latitudes in both hemispheres (see chapter by Pyle, this volume). A reduced stratospheric ozone layer will result in a selective increase at the earth's surface of ultraviolet radiation in the spectral region 280–320 nm (UV-B). The wavelength specificity is due to the absorption coefficient of ozone, which decreases sharply in the short wave UV-B region. Enhanced levels of UV-B radiation may affect the regulatory mechanisms of many plant processes, causing changes in the internal photosynthetic light micro-environment of the leaf, inducing ultrastructural changes, altering morphology and compounding photoinhibition by visible radiation, to name but a few.

The tolerance and avoidance of UV-B stress is manifested in a range of plant strategies, with increased UV-screening compounds being one of the most widely occurring responses. In addition, plants growing in environments of naturally high UV-B irradiances tend to be more tolerant than plants from low irradiance areas (Robberecht & Caldwell, 1986; Ziska, Teramura & Sullivan, 1992). The importance of the distribution within leaves of UV-absorbing pigments can be shown by comparing internal radiation gradients with the pigment concentrations and absorption curves.

Public awareness of the dangers to health of exposure to ultraviolet (UV) light has increased in recent years. Although exposure to UV can have positive effects on humans – sunbathing generally induces a feeling of well-being, partly due to production of natural endorphins, and stimulation of synthesis of vitamin D – the dangers far outweigh the benefits. The most noticeable effect of exposure is sunburn, or erythema; more serious is the increase in risk of skin cancer, and of damage to the eyes, and these symptoms may not surface for several years. At the same time, the public has also become aware that protection from solar UV is provided by ozone in the upper atmosphere, the stratosphere, but that, over recent years, the ozone layer has suffered depletion due to the action of man-made chemicals. The predicted result of this is that more UV radiation from the sun will reach the earth's surface.

Energy from the sun covers the whole electromagnetic spectrum, from short gamma rays (10−5 nm) to long radio waves (103 m). UV light is that region of the spectrum with shorter wavelengths than blue light, between about 400 nm and 250 nm, and this is divided still further into UV-A (400–320 nm); UV-B (320–280 nm) and UV-C (280–250 nm). UV-A does not interact with ozone, since individual photons do not carry enough energy to carry out the necessary photochemical reactions; the energy in UV-B is used in breaking the bonds between oxygen atoms in molecules of ozone, which effectively results in absorption of the UV-B; UV-C is effectively absorbed by ozone/oxygen, and would still be so even under high depletions of ozone.

Introduction

After more than 20 years' research into the effect on plants of elevated levels of UV-B radiation, one might expect it to be a relatively easy matter to predict the impact of future changes in UV-B climate on the growth and yield of important field crops, but this is not the case. Predicting future changes in ground-level UV-B is itself proving very difficult because of uncertainty in trends for future emissions of ozone-destroying chemicals, incomplete knowledge of the atmospheric chemistry involved (see chapters by Webb and by Pyle, this volume) and possible interactions between UV-B and other climate change variables such as CO2 and temperature. Even if future trends were known, recent reviews (for example, Caldwell & Flint, 1994; Fiscus & Booker, 1995) have highlighted the methodological limits of many studies of plant responses to UV-B which make problematic any direct extrapolation to predict likely yield losses at the field-scale. This review of the existing knowledge on field crop responses to UV-B focuses specifically on potential effects on yield. We consider controlled environment, glasshouse and field studies with two questions in mind: (a) will elevated UV-B irradiance alter yields or quality of field-grown crops, and (b) are there beneficial/adaptive effects of UV-B which might be exploited?

Unless otherwise stated, the UV-B quantities discussed below are all in terms of the Caldwell generalised plant action spectrum normalised to 300 nm (PAS300; Caldwell, 1971; Caldwell et al., 1986) as discussed by Holmes (this volume).

Introduction

Penetration of harmful ultraviolet-B (UV-B) radiation (280–320 nm) to the earth's surface is limited by stratospheric ozone. However, ozone depletion, caused by the emission of synthetic chlorofluorocarbons and related compounds, is currently estimated at 4–5% per decade at UK latitudes (Stolarski et al, 1992; Herman, McPeters & Larko, 1993). If the Montreal Protocol continues to be implemented, a possibility that remains uncertain (Greene, 1995; Jordan, 1995), concentrations of chlorine and bromine compounds may reach a maximum in the stratosphere around 1998. Thus, ozone depletion may peak within the next decade, followed by a slow recovery over the next 50 years (Madronich et al, 1995). Despite this more optimistic outlook, other factors such as exaggerated springtime ozone loss in the Arctic due to global warming (Austin, Butchart & Shine, 1992) may need to be considered. In addition, yearly mean ozone depletion figures conceal considerable variation in depletion with season, with maximal losses in late winter and spring (Niu et al., 1992; Herman, McPeters & Larko, 1993; Reinsel et al, 1994), although at that time of year the level of UV-B is relatively low. This evidence suggests that the potential for increasing levels of UV-B over Northern latitudes will remain a problem for many years to come. Many studies on the effects of UV-B on plants have been conducted in controlled environment cabinets or greenhouses, where the levels of UV-A (320–400 nm) and PAR (400–700 nm) may be low compared to the field.

Introduction

The biological consequences of ozone depletion, mediated through an increase in ultraviolet-B (UV-B) radiation, have been cause for concern, prediction and speculation for many years. Estimating the potential effects of ozone depletion involves several steps:

1. Estimating the ozone depletion that might realistically be expected over a given region of the world (this requires assumptions about, for example, compliance with the Montreal Protocol, or not). Alternatively, observed ozone depletions to date can be used to assess the changes already experienced.

2. Calculating changes in UV-B due to changes in ozone. The assumption is that all else remains unchanged and these calculations are usually made for clear-sky conditions. If changes in UV-B irradiances have been observed, then they may be used instead, but they cannot necessarily be attributed solely to changes in ozone.

3. The exposure of biological systems to the available UV-B must be assessed. For plants growing at a single location, this can be assumed to remain unchanged, but for mobile systems (animals, fish, and especially people) adaptive behaviour is possible.

4. The biological (or chemical) results of exposure to the (changed) UV-B must be predicted, based on experiment and observation. Response may depend upon accumulated dose, upon reaching some threshold dose, and upon possible protective mechanisms, for example, the build-up of UV-absorbing pigments (melanin in humans, flavonoids in plants).

Introduction

Small increases in solar ultraviolet-B (UV-B; 280–320 nm) radiation can have substantial effects on the growth and development of many plant species. As a result of a reduction in stratospheric ozone, UV-B radiation has been increasing over Europe for at least a decade, and current evidence points to a gradual increase in incident solar UV-B over Europe at a rate of about 1% per annum (Blumthaler & Ambach, 1990; Ambach & Blumthaler, 1991; WMO Ozone Report Summary, 1994). Over the course of the year, ozone depletion is variable, with the main decrease occurring in late winter and early spring; although total UV-B is much less at this time than during summer, the proportional increase is greatest during spring and is therefore mainly of threat to crops and other plants growing at this time.

Understanding the impacts of this increase on plants requires appropriate action spectra. In approximate terms, an action spectrum indicates the relative effectiveness of different wavelengths of radiation in bringing about a particular response. The relative effectiveness, derived from the action spectrum, can then be multiplied by the irradiance at each wavelength, and summed over the appropriate wavelength range to give the important function, biologically effective radiation. This term will be found throughout this volume; usually it will have been derived from the Caldwell generalised plant action spectrum and be normalised at 300 nm (PAS300). It should be noted that other action spectra are sometimes used.

Introduction

Photosynthesis and photosynthetic productivity in many plant species, although by no means all, can be inhibited by increased exposure to UV-B radiation (Caldwell, Teramura & Tevini, 1989; Tevini & Teramura, 1989; Teramura, Ziska & Sztein, 1991; Tevini, Braun & Fieser, 1991; Middleton & Teramura, 1993; Musil, 1995). To date, there is no consensus for the mechanistic basis of UV-B-induced inhibition of CO2 assimilation in mature leaves. Decreases in Rubisco activity and stomatal conductance have been implicated as factors limiting CO2 assimilation in leaves exposed to elevated levels of UV-B. Prolonged exposure to elevated levels of UV-B has been demonstrated to result in decreases in both Rubisco activity and content (Vu, Allen & Garrard, 1984; Strid, Chow & Anderson, 1990; Jordan et al., 1992; He et al, 1993), and is accompanied by large decreases in the mRNA transcripts of both the large and small subunits of Rubisco (Jordan et al, 1992). Such decreases in Rubisco are consistent with the observed decrease in the leaf carboxylation efficiency, determined from the initial slope of the response of CO2 assimilation to increasing CO2 concentration, when leaves are given supplemental UV-B radiation (Ziska & Teramura, 1992). Exposure to UV-B can also modify the rates of stomatal opening and closing, and reduce the rate of leaf transpiration (Tevini & Teramura, 1989; Middleton & Teramura, 1993; Day & Vogelmann, 1995).

Summary

The ability to isolate angiosperm gametes has opened new experimental avenues, including in vitro fertilization. The use of biotechnological methods such as micromanipulation and single cell culture has led to the technique of in vitro fertilization at the single cell level. In this chapter, a description is given of (a) in vitro fusion techniques using single isolated egg and sperm cell protoplasts of maize and (b) the subsequent development of the fusion product, the zygote, in individual culture. The electrofusion of the gametic protoplasts leads to zygotic embryogenesis and fertile hybrid plants. Furthermore, a nonelectrical alternative technique to fuse isolated higher plant gametes using a fusiogenic medium is given and its relevance to studies of adhesion, recognition, and fusion of these gametes is discussed. The experimental experiences obtained are so far limited to maize. These micromanipulation techniques and their possible application for fundamental and applied studies are described.

Introduction

Biotechnological methods have been applied in addition to sexual crossings in breeding programs for a number of years. For example, cell and tissue culture techniques such as somatic cell genetics, anther and microspore culture, and methods of ovule culture are extensively used. Since the first successful in vitro pollination/fertilization of excised ovules with mature pollen of Papaver somniferum L. was performed (Kanta et al. 1962), techniques of embryo rescue (Stewart 1981) and in vitro pollination/fertilization of flower explants, ovaries, and ovules have been used to overcome cases of self- and cross-incompatibility (for example, Rangaswamy and Shivanna 1967, 1971; Rangaswamy 1977; Zenkteler 1990, 1992). Meiotic and pollen tetrad protoplasts were used in fusion experiments (Ito and Maeda 1973; Deka et al. 1977).

Summary

The pollen longevity of different species varies between minutes and years depending primarily on the taxonomic status of the plant and on abiotic environmental conditions. For a number of agronomically important taxa, including the short-lived graminaceous pollen, special storage conditions are needed to preserve the viability and fertilizing ability of pollen for a long period. Recent sophisticated methods such as nuclear magnetic resonance (NMR) spectrometry, Fourier transform infrared spectroscopy (FTIR), and different ultramicrotechniques for electron microscopy have helped to carry out precise studies on the water regime in pollen, parallel to the molecular changes occurring in membranes during pollen dehydration and rehydration. Cryopreservation seems to be the most efficient method for the long-term preservation (now up to 10 years) of partly dehydrated pollen grains. Beyond the classical role of “pollen banks,” the promising application of the modern in vitro techniques of plant biotechnology (isolation and fusion of reproductive cells, and DNA transformation of artificially produced zygotes and embryos) has opened new, challenging opportunities for germplasm cryopreservation in the near future.

Introduction

The artificial maintenance of the viability and fertilizing ability of pollen over a long period is an important problem from both the theoretical and practical points of view. Historically, the need to retain functional pollen for practical breeding and experimental research has launched detailed studies into pollen morphology, genetics, and physiology. As early as 1885, William King referred to the necessity for learning how to store pollen and stated, “Nothing could tend more to the speedy termination of an experiment than that we had control over the supply of pollen, so that we might use it when and where convenient to ourselves.”

Summary

Under the appropriate in vitro culture conditions, anthers and isolated microspores of higher plants develop haploid embryos by a process referred to as androgenesis. Embryo development can be a direct recapitulation of the developmental stages characteristic of zygotic embryos, or it can be preceded by a callus stage.

There are a number of factors governing pollen embryogenesis, but genotype, donor plant physiology, stage of microspore development, and in vitro culture conditions are the most important. Some characteristics of embryogenie microspores have been identified, and late uninucleate to early binucleate cells are the most responsive. Under inductive culture conditions, uninucleate microspores divide symmetrically to initiate embryogenesis, whereas binucleate pollen exhibits sustained cell division of the vegetative or generative cell. High carbohydrate levels and an initial period at high temperature are conducive to pollen embryogenesis in some species.

Pollen embryos are developmentally similar to zygotic embryos and, under the appropriate culture conditions, they can mature and accumulate seed-specific storage products in a comparable manner. Such embryos develop directly into plants, although plants may arise from secondary structures. Double haploid plants are produced through chromosome doubling techniques. These homozygous plants are useful in plant breeding and genetic studies. In addition, haploid embryos are used in mutant isolation, gene transfer, studies of storage product biochemistry, and physiological aspects of embryo maturation.

Introduction

The occurrence of haploid embryos in plants was first reported by Blakeslee et al. in 1922. Other reports indicated the recovery of haploids, probably pollen derived, as a consequence of interspecific hybridization and embryo development without fertilization (Kostoff 1934).

Summary

The angiosperm microgametophyte displays a complex genetic program during its development. There appear to be two main classes of genes expressed during pollen development, with the transition occurring roughly at microspore mitosis. Although the earlier class of genes is presumed to be associated with immature microspore formation, the later genes probably represent those associated with pollen maturation, germination, and tube growth. This is supported by the observation that several pollen-specific clones of the late class have been identified whose sequences are similar to enzymes associated with cell wall metabolism, as well as many cy to skeleton genes, the products of which are clearly necessary for pollen tube growth.

The genetic promoter elements required for a pollen response continue to elude our understanding, but the seemingly unrestricted interchangeability of pollen promoters among a wide range of host plants argues for some kind of universal pollen element within them. Numerous pollen-specific genes have been isolated recently, and when their promoters are studied in detail, it might be easier to identify common elements in the promoters. Eventually, characterization of genes and promoters involved in key regulatory processes in pollen should provide insights into the nature of haploid gene expression and its relation to that of the diploid plant. Findings of this nature should point the way toward practical applications of pollen molecular biology in the areas of plant breeding, biotechnology, and basic science.

Introduction

A pollen grain represents the male portion of the gametophytic stage in the angiosperm life cycle. Following the completion of microsporogenesis and microgametogenesis, the pollen grain is only a three-celled organism.