Shortterm fate of sulphate acquired by the root

Plants normally acquire their S as sulphate from soil via one or more high affinity sulphate transport proteins, which occur exclusively in root parenchyma. Very little of the sulphate acquired by root parenchyma cells is normally assimilated in the root (Cram 1983b). Thus, the metabolic activity of the root is not likely to be important in regulating sulphate uptake in these cells. In S-adequate plants, the sulphate acquired by root cells is both sequestered in root vacuoles and exported in the xylem but when plants are transferred to sulphate-free medium most of the label stored in root vacuoles is transferred into the xylem. Therefore the short-term distribution of sulphate is very dependent upon the S status of the plant. Collectively, these processes and the sites at which they occur have important implications for the control of sulphate assimilation in whole plants and provide important grounds for suspecting that glutathione, which is the major form of organic S in phloem (Rennenberg 1982, Lappartient and Touraine 1996), serves as the most important long-distance signalling metabolite for the control of sulphate uptake in the root.

The withdrawal of sulphate from root parenchyma of plants grown at very low S occurs passively in response to a decrease in the concentration of sulphate in the cytoplasm as sulphate is conducted from the root to the shoot in the xylem as a result of transpiration activity (Clarkson et al. 1993, Bell et al. 1995). If the activity of the sulphate uptake mechanism in root cells is controlled by the concentration of sulphate in root cytoplasm then the conduction of sulphate from root cytoplasm into the xylem would lead directly to enhanced sulphate uptake. However, there is no direct evidence linking the activity of sulphate uptake to the concentration of sulphate in the cytoplasm. Moreover, if such a system did exist, it would be unresponsive to the requirement for S at the sites of demand in the shoot.

The conduction of sulphate from root cytoplasm into the xylem is thought to be controlled by organic S. Herschbach and Rennenberg (1991) found that exogenous applications of glutathione and cysteine strongly inhibit movement of sulphate from root parenchyma into the xylem. This is consistent with the hypothesis proposed by Rennenberg and Lamoureux (1990) that glutathione acts as a signal for relaying the S status of the shoot to the root. In this way, glutathione would have two effects which could be mediated in the root either directly or, since plants contain mechanisms for the interconversion of glutathione and cysteine, indirectly via cysteine. One effect concerns control of the high affinity sulphate transporter(s); the other concerns the loading of sulphate into the xylem. As argued before, there are theoretical reasons for suspecting that the glutathione response is mediated via cysteine since the cysteine pool is kept under tighter control and Rennenberg (1982, 1984) has proposed that glutathione can be considered as a reserve of reduced S.

Grain development in generative plants is associated with the production of storage proteins thereby necessitating the import of S by developing grains. In theory, S could be acquired directly from exogenous sources such as soil sulphate but this presupposes that generative plants have the capacity to acquire exogenous sulphate and conduct it in the transpiration stream, directly or indirectly, to developing grains. In cereals, the leaves are essentially fully expanded at this time (MacKown et al. 1992) so that there would be little competition for sulphate acquired in this way. However, direct utilization of exogenous sulphate for grain growth presupposes that developing grains possess mechanisms for the reductive assimilation of sulphate-S into the protein S-amino acids.

In practice, the acquisition of exogenous sources of inorganic nutrients, including sulphate, during generative growth is very dependent on water availability. Plants can acquire exogenous nutrients, including sulphate, under very moist or irrigated conditions (Smith and Lang 1988, Smith and Whitfield 1990, Larsson et al. 1991). Moreover, in generative wheat, the sulphate-S acquired in this way can be assimilated into grain storage proteins (Fitzgerald 1997). However, acquisition of exogenous S for grain growth is relatively unimportant in most non-irrigated regions of the world when drying soils at the time of generative growth restrict nutrient uptake (Gregory et al. 1979, Smith and Whitfield 1990). Thus, under these conditions, as discussed below, generative plants must acquire S for grain growth from the endogenous sources acquired during generative growth.

Short-term fate of sulphate arriving in the shoot from the root

In S-adequate vegetative plants, newly acquired S is delivered predominantly to the expanding leaves; fully mature leaves are quantitatively unimportant sinks for newly acquired sulphate (Smith and Lang 1988, Adiputra and Anderson 1992, Sunarpi and Anderson 1996). Since xylem transport directs movement of materials into leaves in proportion to their leaf area, this implies that plants must have a mechanism for transferring sulphate from the xylem into the phloem so that it is delivered specifically to the expanding leaves (Smith and Lang 1988). Consistent with this, Smith and Lang (1988) found that when plants were treated with m-chlorophenylhydrazone, an inhibitor of phloem loading, sulphate was distributed to leaves in proportion to their leaf area. In stylo, the only sulphate transporter found in shoot material is SHST3, which has low affinity for sulphate.

Expanding leaves have an active sulphate assimilation pathway (Schmutz and Brunold 1982, Brunold 1993). For example, ATP sulphurylase is reportedly very active in expanding leaves but mature leaves exhibit very low activity (Adams and Rinne 1969). Much of the sulphate delivered to expanding leaves is assimilated into S-amino acids and incorporated into constitutive proteins as the leaf grows. Expanding leaves also contain high concentrations of glutathione although it is not clear how much of this is imported from other plant parts and how much is synthesized de novo from sulphate within the leaf.

In S-adequate plants, sulphate accumulates in leaf vacuoles. Since mature leaves exhibit very low net synthesis of protein and do not assimilate sulphate (Adams and Rinne 1969, Schmutz and Brunold 1982, von Arb and Brunold 1986, Brunold 1993), then leaf vacuolar sulphate is a potential reserve of sulphate which, in view of the slow rates of passive sulphate efflux across the tonoplast (Bell et al. 1994), can be slowly mobilized in the event of a decrease in the sulphate concentration in the cytoplasm (e.g. S stress). Consistent with this, the mature leaves of plants which have been grown under S-limiting conditions do not synthesize additional protein when the plants are provided with adequate S although the sulphate content of the leaves might rise considerably (Smith and Lang 1988, Dietz 1989). Sul-phate-S in leaf vacuoles effluxes slowly in vegetative plants. Consistent with this, pulse chase experiments with S-adequate plants indicate that a large proportion of the S acquired by a leaf while it is expanding is not incorporated into the insoluble (protein) fraction and is subsequently exported from the soluble fraction over a very extended period of time (Adiputra and Anderson 1992). The rate of export of S from leaves of vegetative plants does not appear to be especially responsive to the imposition of S deficiency (Adiputra and Anderson 1995, Sunarpi and Anderson 1996) but this is not the case in generative plants (Fitzgerald et al. 1999a) where developing grains impose a very strong demand for S.

The discussion above has focussed on the redistribution of S that is delivered in the xylem to mature leaves. However, it should be noted that redistribution of S from the leaves of vegetative cereals and soybean is evident well before they attain full expansion (Adiputra and Anderson 1992, Sunarpi and Anderson 1996). Given that expanding leaves actively assimilate sulphate into cysteine and glutathione then the possibility that a significant amount of S found in the phloem originates from these leaves as glutathione rather than as sulphate (as is the case in mature leaves) needs to be addressed.

Transport within the shoot and recycling via the root

Since expanding leaves are the principal site for the assimilation of sulphate into cysteine and glutathione (Brunold 1993) and roots are not quantitatively active in this way (Cram 1983b), then leaves must be an important source for the long distance transport of organic S to sites that do not actively assimilate sulphate, both within the shoot itself and to the root. This must involve the long distance transport of glutathione since the phloem must act as the conduit for the transport of organic S from the shoot to the root and glutathione is the major form of organic S in phloem (Lappartient and Touraine 1996). This has several implications. Most importantly, it provides a mechanism for conveying a signal about the S status of the shoot to the root which can be used to regulate the activity of the sulphate uptake mechanism and also influence the loading of sulphate from the cytoplasm of root parenchyma into the xylem and hence movement of sulphate from root vacuoles via the cytoplasm into the xylem as proposed by Rennenberg et al. (1989). Second, it implies a phloem unloading mechanism in the root and raises the question of the eventual fate of the glutathione in the root. Presumably it can serve as a source of reduced S for the formation of essential S-compounds (e.g. cysteine and methionine for incorporation into proteins) required for root growth and maintenance. Another possibility is that glu-tathione is recycled to the shoot in the transpiration stream where it is perhaps reloaded into the phloem a second time (S recycling hypothesis), a process that would involve phloem to xylem transfer in the root.

Evidence for S recycling of endogenous S (Figure 5) has been obtained using a split-root technique (Cooper and Clarkson 1989, Larsson et al. 1991). In wheat, S recycles as sulphate and recycling S does not mix with other S pools within the root (Larsson et al. 1991). However, definitive demonstrations that endogenous glutathione, emanating from the shoot, also recycles still provides a formidable technical challenge.

Since sulphate recycles, this raises the question whether sulphate arriving in the root in the phloem could act as a signal for regulating sulphate uptake in the root. To date, there is no experimental evidence for excluding this possibility.

In generative plants grown with adequate water, exogenous sulphate is the main form of S for the synthesis of grain proteins (Fitzgerald et al. 1999b). However, under normal field conditions, without addition of abundant water, the main endogenous sources of soluble S which are used for grain growth are sulphate and glutathione acquired/formed in vegetative tissues from S during vegetative growth. Sulphate occurs in the vacuoles of roots, leaves and stems and diffuses passively into the cytoplasm when the cytoplasmic concentration falls below a critical concentration (Clarkson et al. 1983). To reach the grain, sulphate has to be loaded into the phloem since redistribution via the xylem would direct sulphate to transpiring tissues. In any event, the apoplastic discontinuity to the endosperm cavity (Zee and O'Brien 1970) from which metabolites are recruited into the developing endosperm (Ugalde and Jenner 1990a) would prevent direct delivery of sulphate via the xylem. As discussed below, developing grains appear to have the capacity to incorporate sulphate-S into protein-S so therefore they must have mechanisms for recruiting sulphate from the endosperm cavity. Sulphate occurs in the developing grains of wheat plants grown under S-sufficient conditions (Roberts and Koehler 1966) and, under these conditions, is a quantitatively important source of S for grain development (Fitzgerald 1997). This is consistent with earlier observations which indicate that the amount of S delivered as cysteine and methionine is far too small to account for the S occurring in the S-amino acids in grain proteins (Fisher and MacNicol 1986, Blumenthal et al. 1990, Ugalde and Jenner 1990b). However, in plants grown with inadequate S, the amount of endogenous sulphateS available for redistribution to developing grains is negligible (Fitzgerald et al. 1999b) and under these conditions, the S required for grain growth must come from other endogenous sources.

Glutathione is a quantitatively important form in which S is imported into developing grains (Fitzgerald 1997). Indeed, in plants grown at low S during vegetative growth, glutathione accounts for about 86 % of the soluble S in the endosperm cavity suggesting that it is the main form in which S is transported in these plants. The transported glutathione is probably derived from at least two sources. One probably concerns the free glutathione present as a constitutive metabolite in chloroplasts. Presumably glutathione is recruited from this pool during generative growth, perhaps by some form of regulated senescence or enhanced membrane permeability to glutathione. The other likely source is protein-S.

Role of glutathione in the remobilization of protein-S in vegetative plants

Remobilization of protein-S can be readily demonstrated in germinating seeds. In soybean, for example, the amount of insoluble S in the cotyledons declines to very low levels as germination proceeds (Sunarpi and Anderson 1995). This is accompanied by a transitory rise in the concentration of soluble S in the cotyledons. Subsequently, the S lost from the insoluble fraction in the cotyledon is quantitatively recovered in the growing seedling (Sunarpi and Anderson 1995). Similarly, in germinating barley, the amount of insoluble S in the endosperm declines during germination and the amount of glu-

tathione and soluble (free) methionine increases sharply (Imsic and Anderson, unpublished data). The production of glutathione implies that germinating barley has a mechanism for mobilising S from storage proteins into cys-teine and/or methionine (or products derived from them) and incorporating the S into the cysteinyl residues of glutathione. Presumably this involves processes similar to those depicted in Figure 2. This raises interesting questions about the control of glutathione synthesis during seed germination and its coordination with protein hydrolysis in germinating seeds.

N stress promotes a marked increase in the export of protein-N from mature leaves to developing leaves (Mei and Thimann 1984). Conversely, mobilization of protein-S from mature leaves of vegetative soybean plants grown at high N is not especially responsive to S deficiency (Sunarpi and Anderson 1996). However, the loss of insoluble S (protein-S) from mature leaves of vegetative soybean grown at low S is very sensitive to N stress (Sunarpi and Anderson 1997b). The loss of insoluble S is accompanied by a slightly greater proportional loss of insoluble N implying the preferential hydrolysis of S-poor proteins from mature leaves. These data are not inconsistent with the hydrolysis of vegetative storage proteins; these proteins, which are known to occur in soybean leaves, are relatively poor in S amino acids, and are hydrolysed in response to low N (Staswick 1994). The loss of protein S from mature leaves of N-stressed soybean plants is accompanied by a minor increase in the concentration of glutathione in these leaves and a very large increase in glutathione in the young developing leaves at the shoot apex (Sunarpi and Anderson 1997b). These data are consistent with N-stress induced hydrolysis of protein-S and synthesis of glutathione in the mature leaves linked to transport of glutathione to the young leaves at the shoot apex where it accumulates, presumably because the level of N nutrition is too low to support the synthesis of constitutive proteins required for new growth. Perhaps this would prove a suitable system for examining whether glu-tathione provides the signal for arresting sulphate uptake in the root.

Role of glutathione in the remobilization of protein-S to developing grains

It is well known that the level of leaf protein falls during grain growth, especially in N-stressed plants (Neales et al. 1963, Dalling et al. 1976, Dal-ling and Simpson 1981, Simpson et al. 1983, MacKown et al. 1992). Largely for this reason, protein-S has long been thought to be an important precursor of the S imported into developing grains since hydrolysis of leaf protein would be expected to release soluble forms of S as well as N. Recently, this hypothesis has been examined by several groups although the effect of N nutrition on the mobilization of leaf protein-S for grain growth has yet to be examined. Generative cereals exhibit strong mobilization of protein-S in response to S stress (Fitzgerald et al. 1999a,b). Indeed, in plants grown at low S during vegetative growth the loss of insoluble S from vegetative tissues quantitatively accounts for the gain of protein-S in the developing grains. Fitzgerald (1997) examined the S composition and turnover of the contents of the rachis, endosperm cavity, and endosperm and calculated that glutathione accounted for about 84 % of the S imported into developing grains in plants supplied with inadequate S during vegetative growth. The corresponding figure for plants grown with adequate S during vegetative growth was 37 %, most of the balance being supplied by sulphate. Since leaf protein-S accounted for all of the S imported into the grains in the plants grown with inadequate S, this implies that almost all of the remobilized leaf protein-S was metabolized to glutathione, again emphasising the importance of processes such as those shown in Figure 2.

The above data draw attention to the importance of glutathione synthesis in senescing leaves and cotyledons as a way of mobilising S from proteins. Little is known about the types of proteins from which S is recruited and this draws attention to the fact that leaves and the storage tissues of seeds contain specific storage proteins which differ greatly in their S content as shown for the storage proteins of wheat endosperm (Table 2). In addition to the vegetative storage proteins described above, leaves contain very large amounts of the functional protein ribulose bisphosphate carboxylase, which is also subject to proteolysis (Vierstra 1993). The mobilization of S from proteins such as these and the pathway for the inferred incorporation of methionine-S into glutathione and its regulation remain as challenges for the future. Another matter still to be addressed is the mechanism of glutathione uptake from the endosperm cavity and its presumed metabolism to cysteine and methionine in developing grains.

Table 2. Cysteine (Cys) and methionine (Met) contents of some major wheat storage proteins.

Protein No. of amino acid Composition residues

Table 2. Cysteine (Cys) and methionine (Met) contents of some major wheat storage proteins.

Protein No. of amino acid Composition residues

Total

Cys

Met

%N

%S

HMW Glutenin1

842

4

3

18.3

0.61

LMW Glutenin2

354

8

6

18.9

1.10

a/ß-Gliadin3

243

5

2

17.9

0.87

y-Gliadin4

292

9

6

17.3

1.45

(ü-Gliadin5

330

0

0

¡8.5

0.00

Sources: 1, Sugiyama et at. 1985; 2, Pitts et al. 1988; 3, Garcia-Maroto et al. 1990; 4, Rafalski 1986; 5, Castle and Randall 1987.

Sources: 1, Sugiyama et at. 1985; 2, Pitts et al. 1988; 3, Garcia-Maroto et al. 1990; 4, Rafalski 1986; 5, Castle and Randall 1987.

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