9.2.2  Diverse roles for plant hormones

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Before we consider details of the final consequences of hormone signalling pathways, it is helpful to think broadly about the roles of hormones in enabling organised plant development and efficient responses to alterations in the environment. The range of functions of plant hormones and responses to them can be bewildering, but most roles can be grouped under two general headings: organisers and mediators.

(a)  Organisers

Organisers primarily define the basic framework of a plant’s axial structure. This includes channelling of cells into particular pathways of differentiation depending on their location within the plant. Plants tend to, even need to, maintain a balance between shoot and root development. The two systems are interdependent, so damage or loss of one upsets that balance and necessitates some developmental adjustment. We do not know exactly how complex differentiation pathways are regulated, but it seems likely that a relatively small number of primary signals are needed, with each controlling a whole suite of characters. In Section 9.2.3, we see how such systems can operate at the level of gene expression.

Auxin–cytokinin balance: opposing directional flows of active signal

Tsui Sachs and Kenneth Thimann in 1967 proposed that shoot apical dominance is governed by auxin–cytokinin balance. Evidence came mainly from polar basipetal transport of shoot tip auxin and responses to applied auxins and cytokinins: auxin supplied to a decapitated shoot stump suppresses the normal lateral bud growth response, but cytokinin supplied directly to lateral buds promotes outgrowth of intact plants. More recent studies on transgenic plants and branching mutants suggest that there are probably other regulatory signals in addition to auxin and cytokinin (see Case study 9.1). Overall, however, this simple theory, along with auxin and cytokinin responses in tissue cultures (see Section 10.2 and Figure 10.23), gives us the foundations of control of root:shoot development ratios, and enables comprehension of plant developmental homoeostasis. This balancing act comprises several developmental elements, but all potentially trace back to auxin:cytokinin concentration ratios. Even in intact plants, shoot branching is limited if root growth is poor or if roots are stressed. Conversely, a vigorous root system depends on carbon supply from shoot photosynthesis. Superficially, root:shoot balance appears resource limited, but the role of hormones overlays an ability to signal in advance of crisis, and enables stochastic (modular) adjustment of units of plant development: number of active shoot branches and number of lateral roots, in addition to modification of the growth rate of each. Mechanical damage, whether removal of just a shoot apex, or cutting off a stem at ground level, may invoke similar hormone-driven responses. Shoot apex replacement is rapidly achieved by outgrowth of an existing lateral meristem, in theory stimulated by auxin depletion and cytokinin accumulation around the top of a cut stem. A tree stump lacking reserve buds may still possess active cambium cells, which can respond to the same cytokinin enhancement by initiating rapid cell division and subsequent shoot organogenesis (see Section 10.2.2). A stem cutting continues to transport auxin to the cut base, but lacks its normal cytokinin supply from the roots. This high auxin:cytokinin ratio stimulates cell activation leading to adventitious root organisation, and in commercial propagation is accelerated by supplying additional auxin in rooting powders and solutions. In addition, there are strong links between cytokinins and delay of senescence (Gan and Amasino 1996; Wang et al. 1997). A plant with damaged, diseased, water-stressed or mineral-deficient roots will export less cytokinin in the xylem, one con-sequence being premature leaf senescence usually from the stem base upwards.

Seed-derived hormones regulate pattern of fruit development

Although fruit and seed tissues are genetically different — the former is parental, the latter is progeny — the two develop in a coordinated manner. In most species, if ovules are not fertilised or the embryo aborts, fruit tissues stop growing and are usually shed prematurely. Because seeds represent the next generation, it makes little sense for a plant to continue investing resources in a package that contains no propagules. Exceptions to this are parthenocarpic (seedless) fruits, some of which have genetic causes, and others which are induced chemically by application of growth-promoting hormones: auxin, gibberellin, cytokinin or combinations of these. Parthenocarpic fruit are prized by humans and include seedless or semi-seedless commercial varieties of grape (see frontispiece to Chapter 11), citrus, banana, watermelon, pineapple and lychee. The relationship between seed and fruit growth appears to be driven by hormones synthesised in the developing endosperm and embryo, and is neatly illustrated by the relationship between auxin levels and fruit growth rates in blackcurrant (Figure 9.6).

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Figure 9.6 During seed and fruit development in many species there is a phase of endosperm development followed by embryo growth. These two tissues are thought to be the source of hormones that promote fruit growth. Here, the two rapid phases during the double sigmoid fruit growth curve of blackcurrant (Ribes nigra) coincide with peaks of IAA content (solid circles) and with maximal rates of endosperm then embryo development (open circles).

(Based on Wright)

(b)  Mediators

The second broad role of hormones is as mediators of environ-mental signals, often stress factors, which lead to modification of physiology and patterns of development. For example, as discussed in the preceding chapter, photoperiod perception in leaves leads to flowering at the shoot apex. A light signal is translated into a chemical one. The route of signal trans-mission from leaf to apex appears to be in the phloem sap, and although we do not know of a universal ‘florigen’ hormone, part of the signal in some species may be specific types of gibberellin (Evans et al. 1994b, c). Gibberellins also have a wider role in mediating some types of phytochrome responses, such as stem elongation in long-day plant rosette species (Wu et al. 1996). Entry into and exit from dormancy often depends on external inputs such as fluctuation in water availability: some responses to dehydration during seed maturation and imbibition during germination are mediated by ABA and gibberellins, respectively. Low temperature is another factor which can break endodormancy or induce flowering, and may be mediated by hormones such as gibberellins and cytokinins.

ABA as a mediator of water status information

The role of ABA in transmitting information about plant water status was discovered in the early 1970s. Here, we take a detailed look at one of the best studied of all ‘mediator’ roles of hormones. When water loss from leaves is accelerated by exposing them to a stream of warm air, ABA concentration rises dramatically, by about 10- to 50-fold and stomata close (Zeevaart 1980). Similarly, a low concentration of ABA supplied exogenously to excised leaves via the transpiration stream induces stomatal closure. It was concluded that ABA synthesis in leaves, induced by water stress, is the cause of stomatal closure. Further evidence for the involvement of ABA in stomatal regulation came from studies of ABA-deficient mutants such as flacca in tomato, wilty in pea and droopy in potato, which wilt rapidly when exposed to only mildly stressful conditions but regain a normal phenotype if treated with ABA.

Synthesis of ABA in wilting leaves is enhanced as turgor approaches zero. Conjugated forms of ABA such as the b-glucoside can be present in leaves at quite high concentration and represent a potential source of free ABA, but actually appear quite stable and do not break down under stress. Therefore de novo synthesis of ABA during stressful conditions is responsible for stomatal closure, and acts as a protective mechanism against the potentially damaging effects of water loss. ABA-induced stomatal closure pre-empts hydraulically driven stomatal closure which would eventually occur as stomatal guard cells lose turgor. Hydraulically driven closure occurs far later than closure induced by ABA and usually occurs too late to prevent excessive and damaging levels of water deficit.

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Figure 9.7 ((a), (b), (c)) Response of grapevines to hydraulic and non-hydraulic signals during water stress applied to split root systems. Root systems can be split vertically (surface v. deep) or horizontally (left-right, as in this experiment (d)) and different water stresses applied to each half. If one half is kept well watered, this is sufficient to maintain normal shoot water status and no leaf water potential difference is detected (solid circles) compared with control fully watered plants (open circles) (b). If the other half root system is dry, any drought signal induced may be transmitted to the shoot. A prime candidate for this root signal is ABA, which is detected either chemically (c), or by its effects on stomatal aperture (a). In many species, root-generated ABA can cause stomatal closure in the absence of any water deficit in the shoot. This is interpreted as an early warning system, enabling plants to reduce water use under imminent drought conditions. In nature, during periods without rain, surface roots would normally become dry before deep roots.

(Diagram courtesy B.R. Loveys)

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Figure 9.7 (d)

Stomatal closure in response to increased levels of foliar ABA provides a solution to one of the major problems faced by mesophytic plants, that is, the compromise between maintaining sufficient gas exchange to satisfy the CO2 requirements for carbon assimilation but at the same time limiting water loss when conditions become unfavourable. However, this rather simple interpretation of plant response to stress is not the whole story. For example, water-stressed plants can have leaf water potentials which are similar to or even higher than those of well-watered plants and yet the stomata are fully closed (Figure 9.7). Shoot extension and leaf expansion are also highly sensitive to stressful conditions but they are not always accompanied by low leaf water potentials. Clearly, ABA synthesis in leaves is not the only process occurring during water stress.

Roots as a source of ABA

Part of the answer to this puzzle came from experiments using plants with divided root systems. For example, if a piece of grapevine cane bearing six or seven nodes with dormant buds is sawn along its length from the base for about two internodes, it is possible to induce root formation on each of the two halves. These split root systems can be planted in separate pots or in the field, which allows independent manipulation of the soil moisture status of each root (Figure 9.7d). It has long been known that xylem sap contains ABA (Figure 9.2), and that increased ABA in droughted roots might thus be transmitted to the leaves (Davies and Zhang 1991). However, simply drying the roots of a plant with a single root system affects the water relations of the whole plant and it is then difficult to distinguish the effects of lowered leaf water potentials from the effects of root-derived chemical signals. Split-root plants allow study of the effects of drying soil without the complications of changed water relations because the soil around one root system is maintained fully watered. This root system supplies as much water as the canopy needs. Normally, the second root system is then dried and the effects of any chemical signals studied. Here we show the effect of withholding water from one root system of twin root grapevines (Figure 9.7). Stomatal conductance fell rapidly, and within 8 d was only 22% of that in fully watered control vines yet water potential of the leaves remained unchanged. Leaf ABA content also changed in response to partial root drying. When conductance was at its lowest, ABA levels had doubled when compared with fully watered controls. ABA levels returned to control values 10 d after rewatering the dry pot, but conductance took somewhat longer to recover.

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Figure 9.8 Large fluctuations are often seen in ABA concentrations in xylem sap moving from root to shoot. In some species such as maize (but not others such as wheat and apricot; see Figure 9.9), drought-induced stomatal closure can be accounted for entirely by the increased amount of ABA signal. In this experiment, increased ABA was generated by withholding water for up to 20d. The error bars on each point are standard deviations and indicate the range of both the increase in ABA content and the effect on stomatal conductance. The open circle shows the xylem ABA concentration and leaf conductance resulting from feeding 10-5 M ABA to part of the root system.

(Based on Zhang and Davies 1990; reproduced with permission of Blackwell Science)

The leaves did not receive any hydraulic (water deficit) signal which may have initiated local ABA synthesis. We conclude that the drying roots produced a chemical signal which is transported to the leaves in the transpiration stream and which induces stomatal closure. The chemical signal is most likely to be ABA. Zhang and Davies (1990) took a direct approach by supplying different solutions to excised maize leaves and measuring stomatal conductance (Figure 9.8). They tested xylem sap from well-watered plants, sap from stressed plants, sap from which most of the ABA had been removed by passage through a column containing anti-ABA antibodies and a series of synthetic ABA solutions. In every case, the anti-transpirant activity of each solution was explicable in terms of its ABA content. Further evidence that it is a closing stimulus arising from drying roots and not a lack of an opening stimulus comes from the observation that stomata re-open after drying roots are excised.

Experiments like this help us understand how plants in the field deal with everyday fluctuations in soil water, sustained drought and other environmental conditions. Surface layers of soil, which usually have the highest root densities, dry first and roots in this zone will be stimulated to send enhanced ABA signals to the leaves, slowing transpiration and thus providing the first indication that soil conditions are not ideal. At this time, the deeper roots may still have access to water and so no hydraulic signal has been generated. Such ABA signalling from roots does not result in large increases in leaf ABA and that which does accumulate is soon dissipated through metabolism and translocation once the dry root signal is removed. This enables leaves to monitor continuously root water stress and to adjust stomatal apertures according to distant and local water availability. If drought continues and more of the soil profile dries, then leaf water potentials will fall and trigger new synthesis of ABA in leaves, reinforcing and extending the stomatal closure already set in train by the roots. The large increases in ABA which then result may have more far-reaching consequences because expression of certain genes is regulated by ABA (see Section 10.3). Some of these have sequences which are predicted to confer heat stability to their resulting protein products.

Unexpected relationships between conductance and ABA content

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Figure 9.9 In an unusual response, stomatal conductance was postively correlated with leaf ABA concentration and not inversely correlated as expected. This study used apricot plants, which are able to osmoregulate during drought and thereby maintain cell turgor and hence open stomata. The increase in ABA is probably the result of increased supply from the roots during drought, but in this case the ABA does not result in stomatal closure.

(Based on Loveys et al. 1987)

The picture of ABA derived from roots causing reduced stomatal conductance during periods of water deficit is true in many, but not all, cases. For example, this correlation does not hold in apricot which instead can osmoregulate and thereby maintain stomatal opening. In a study comparing the drought responses of grapevine, which largely conforms to the normal model, with apricot, another drought-tolerant plant often grown in close proximity to grapevine, xylem ABA content in apricot was only about 5% of that needed to induce stomatal closure. Moreover, ABA levels in leaves showed a positive relationship with stomatal conductance, contrary to normal expectations (Figure 9.9). ABA was accumulating with increased transpiration but was having no effect on stomatal conductance. Unlike grapevine, which operates over a fairly narrow range of leaf water potentials, apricot leaf water potential fell progressively throughout the season, yet stomata remained open. The key to these apparently anomalous results was that leaf osmotic potential fell along with the water potential, thus maintaining leaf turgor and stomatal opening. By the end of the growing season, sorbitol, the major organic osmoticum in apricot leaves, had accumulated to a concentration of 400 mM. Apricot uses osmotic adjustment to protect itself during drought and ABA appears to play little part. Indeed, it was found that even when apricot leaves were deliberately wilted, their ability to synthesise ABA was almost totally eliminated when sorbitol concentrations were high (Loveys et al. 1987).

Next, we follow the signalling pathways to their final sites of action, namely modified development and physiology. Essentially, there are two options: direct effects and action through altered gene expression.

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