Subcellular mechanisms of plant response to low water potential

Agricultural Water Management, 7 (1983) 239--248

239

Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

SUBCELLULAR MECHANISMS OF P L A N T R E S P O N S E T O LOW WATER POTENTIAL

J O H N S. B O Y E R

AgriculturalResearch Service, U.S. Department of Agriculture,Department of Botany and Department of Agronomy, Universityof Illinois,289 MorrillHail, 505 S. Goodwin Avenue, Urbana, IL 61801 (U.S.A.) (Accepted 23 November 1983)

ABSTRACT

Boyer, J.S., 1983. Subcellular mechanisms of plant response to low water potential. Agric. Water Manage., 7: 239--248. Understanding the metabolic basis of the resistance of plants to limited water may hasten the development of superior techniques for increasing crop yields. Three promising areas where understanding has recently advanced are osmotic adjustment, photosynthesis, and tolerance to severe desiccation. Osmotic adjustment, i.e., large change in cell solute content, occurs in response to soil water availability and permits plants to maintain growth on limited sou water when growth otherwise would not occur. Grain yields are enhanced in wheat genotypes having superior osmotic adjustment as soils become dry. Osmotic adjustment is controlled by nonsimultaneous changes in the rate of solute uptake and the rate of solute use by cells in response to limited water availability. Photosynthesis is generally inhibited by losses in activity of leaves as well as by losses in viable leaf surface as the availability of soil water decreases. These losses are caused by stomatal closure, decreased chloroplast activity, and factors controlling the persistence of viable leaves. Each of these factors appear to be under metabolic control: stomatal aperture is determined by solute retention by the guard cells; chloroplast activity is altered by concentrations of regulatory ions in the cells; and viable leaf area is controlled by metabolic factors accelerating the normal senescence of the leaves. By contrast, the translocation of photosynthetic products is less sensitive than photosynthesis to limited water supply, which has the effect of maintaining yield by mobilizing stored reserves. The loss of viable leaves contributes only a small amount to this mobilization, and selection against accelerated senescence is probably desireable. Tolerance to severe desiccation can occur in nonsenescing leaves of some native species but not in many crops. The tolerance appears to be associated with the persistence of the nucleus in the leaf cells, because considerable degradation and loss of integrity occurs in all other cell structures. Mechanisms may exist that preserve nuclear integrity when cells are severely desiccated.

INTRODUCTION The response of plants to a limited water supply involves a broad range o f d e v e l o p m e n t a l a n d c e l l u l a r e v e n t s t h a t , f o r n a t i v e s p e c i e s , are a d v a n t a -

240 geous if the plants can produce a few progeny for the next growing season. In agriculture, however, our task is to identify those features that increase yield per unit land area. Many of the features possessed by native species may be of little importance to that goal. Therefore, despite the long time that dry conditions have influenced the course of plant evolution, opportunities should exist to alter plants for the benefit of agriculture with limited water. The developmental and cellular changes that occur in plants in response to limited water supply must be based in metabolism. It is the purpose of this paper to describe several of these features that appear particularly promising for improving agricultural performance of crops and, where possible, to relate them to crop yield with limited water supply. OSMOTIC ADJUSTMENT The high concentration of solutes found in plant cells creates the ultimate driving force, i.e. osmotic potential, that brings water into the plant from the soil. Osmotic adjustment involves the accumulation of solutes in sufficient quantity to change the osmotic potential. Within a particular tissue, the osmotic potential depends on the balance between the rate of solute accumulation and the rate of solute use by the cells. Thus, concentrations increase when accumulation exceeds use but decrease when the reverse occurs. By far the largest flux of solutes entering cells is photosynthate and the largest use of solutes consists of respiration as well as the polymerization of photosynthate to form the protein, fats, cell walls, nucleic acids, and other complex constituents of cells. Inorganic ions accumulated from the soil also contribute significantly to the osmotic potential. Potassium, and to a lesser extent nitrate and chloride, can accumulate in osmotically significant amounts. In salinized plants, sodium may be present. Indeed, increases in sodium chloride concentrations were thought to account for most of the osmotic adjustment in cells of salinized plants (Eaton, 1927; Black, 1960; Bernstein, 1961). However, Meyer and Boyer (1972) observed osmotic adjustment in seedlings growing in water deficient vermiculite that contained no significant solutes, and this observation indicated that osmotic adjustment could occur in response to dry soil conditions. Similar findings by Greacen and Oh (1972) and subsequently by others (Cutler et al., 1977; Morgan, 1977; Fereres et al., 1978; Jones and Turner, 1978; Turner et al., 1978a, b; Acevedo et al., 1979; Jones and Rawson, 1979; Munns et al., 1979; Sharp and Davies, 1979; Cutler et al., 1980) confirmed this result with a range of plant species. Attempts have been made to incorporate a large capacity for osmotic adjustment in crops, because osmotic adjustment permits growth to occur under dry conditions that otherwise would be completely inhibitory (Meyer and Boyer, 1972). In Australia, this approach was used to select a drought tolerant wheat that outyielded commercial cultivars by almost 2:1 under

241

dry conditions but yielded as well as the commercial types under favorable conditions (J.M. Morgan and R. Hare, Tamworth, Australia, personal communication, 1981). These investigators used genetic variability for the ability to adjust osmotically to select for the superior cultivar. The solutes involved in osmotic adjustment during drought consist primarily of organic constituents, particularly amino acids, organic acids, and sugars (Cutler et al., 1977; Acevedo et al., 1979; Meyer and Boyer, 1981) for cotton, sorghum, and soybean, although inorganic ions contributed to osmotic adjustment in sunflower (Jones et al., 1980). The accumulation of these solutes may be enough to completely compensate for the altered water status of the root medium. In this case, tissue dehydration is absent and the tissue turgor remains high (Meyer and Boyer, 1972), particularly in regions of rapid cell enlargement, with favorable consequences for growth. How is this osmotic adjustment controlled? Inasmuch as the concentration of cell solute depends on the rates of accumulation and use, control could act on either or both of these processes. A detailed analysis of osmotic adjustment in the stems of soybean seedlings suggests that both processes are involved. Table I shows that dry matter accumulated in the stem for 12 h after the seedling was exposed to vermiculite having a low water potential (--0.3 MPa, obtained by maintaining a low water content). Most of this dry matter accumulated in the elongating region of the stem, since within 4 h after exposure to the dry vermiculite the production of TABLEI

Rates of dry matter accumulation in stems (hypocotyls) of soybean seedlings growing in the dark at high humidity after being transplanted to vermiculite having low water content Time after Entire stem Stem elongating Stem mature transplanting (rag h -~ per stem) region region (h)

0 4 8 12 16 20 24

0.25 0.25 0.16 0.07 0.0 0.0 0.0

Osmotic potential in stem elongating

(mg h -I per section)

(mg h -I per region mature region) (MPa)

-0.13 0.20 0.13 0.07 0.0 0.0 0.0

0.38 0.05 0.03 0.0 0.0 0.0 0.0

--0.74 -0.82 --1.05 --1.08 --1.12 --1.13 --1.13

Controls were transplanted to vermiculite of high water content. Rates of dry matter accumulation in the controls were 0.25 m g h -I per stern and constant. For the elongating region, the rate was --0.13 m g h -I per section for the first 3 h and 0.0 thereafter. For the mature region, the rate was 0.38 m g h -~ per mature region for the first3 h and 0.25 m g h -~ per mature region thereafter.The osmotic potential of the elongating region of the controls was -0.74 M P a and constant. For details,see Meyer and Boyer (1981).

242 mature tissue by the stem was inhibited. The production of mature tissue is a measure of the rate of biosynthesis for the stem, and therefore the accumulation of dry matter by the stem exceeded the utilization of dry matter for biosynthesis between 4 and 12 h after transplanting. As a consequence, dry matter unused for biosynthesis 'piled up' in the elongating region and this region became heavier. This result was in contrast to the controls, which used dry matter for production of mature tissue at a rate a b o u t equal to the accumulation of dry matter by the stem. The behavior of the seedlings in dry vermiculite suggests that the control of osmotic adjustment occurs at two sites: (a) there is cellular control of the rate of biosynthesis, which in turn determines solute use (we may disregard respiratory effects in this analysis because respiratory activity accounts for equivalent components of solute uptake and use, and the net effect is zero); (b) there is control of the rate of uptake of solute by the cell, perhaps at the plasmalemma. If changes in these two control sites are not simultaneous, solute accumulates or is depleted from the solute pools in the cells. In soybean stems, osmotic adjustment involved decreased biosynthesis rather than increased uptake of solute. However, this behavior may not apply to other tissues. In maize roots, for example, biosynthesis may continue as osmotic adjustment occurs, because dry matter accumulates {Sharp and Davies, 1979). Thus, the regulation of solute uptake and use may differ in different organs. Regardless of the balance between accumulation and utilization, however, control at both sites appears to be central to the process of osmotic adjustment. PHOTOSYNTHESIS

Photosynthesis usually decreases as leaf water potential decreases (Boyer, 1976). Although the decreases in photosynthetic activity were attributed to stomatal closure, recent evidence shows that chloroplast activity is also inhibited (Boyer, 1976). In some plants, these losses in chloroplast activity may be more limiting than stomatal closure at low water potentials (Boyer, 1971; Ackerson et al., 1977). Stomatal closure occurs because the guard cells no longer retain high solute concentrations at low water potentials (Ehret and Boyer, 1979). Even when leaf dehydration occurs over times as short as a few minutes, solute release can be observed in the guard cells, and closure follows soon thereafter. Often there is a lag before stomata reopen after water has been resupplied to the plants. This lag is associated with a lag in the accumulation of solute by the guard cells. Therefore, guard cell closure and reopening in response to leaf water potentials are unlikely to be a hydraulic consequence of the change in leaf turgor but rather a result of changes in the osmotic potential necessary for guard cell opening. Thus, stomatal behavior at low water potential is likely to be under some form of metabolic control.

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The losses in chloroplast activity that accompany losses in photosynthesis at low water potentials involve decreases in electron transport and photophosphorylation (Keck and Boyer, 1974) and are associated with changes in conformation of the thylakoid membranes rather than chloroplast degradation or the loss of structural integrity (Fellows and Boyer, 1976). These changes in conformation are in turn associated with conformational changes in subunits of the thylakoid membranes. A recent study (Younis et al., 1979) of the losses in photophosphorylation that take place in chloroplasts exposed to low leaf water potentials showed that coupling factor (ATP synthetase in thylakoid membranes) changed conformation in such a way that the binding of ADP to the enzyme was restricted. Attempts to simulate the effects of low leaf water potentials on chloroplast photophosphorylation were made using Mg2÷ concentrations likely to occur in chloroplasts of cells dehydrated to varying degrees. Table II shows that Mg2÷ concentrations usually present in chloroplasts (1 to 3 mmol 1-1 -- Portis and Heldt, 1976; Portis, 1981) were likely to have increased during leaf dehydration and were associated with an inhibition of photophosphorylation. When Mg2÷ was supplied to isolated chloroplasts at concentrations expected during dehydration, a similar inhibition of photophosphorylation occurred (Table III). Furthermore, this result extended to chloroplast coupling factor assayed as Ca2÷-ATPase (Table III). TABLE II Phosphorylation activity of isolated spinach chloroplasts, approximate water content of spinach leaves, and calculated stromal Mg :÷ concentrations at various leaf water potentials Water potential (MPa)

Photophosphorylation Approximate activity water content (umol h -1 (mg chloroplast) -~) (% of turgid)

Calculated stromal concentration of Mg 2÷ (mmol 1-~)

--0.2

1 060

100

3

--1.5 --2.5

740 475

55 35

6 9

Chloroplasts were isolated from spinach leaves that had been dehydrated to levels shown. Assays were conducted as in Younis et al. (1979). Stromal Mg :÷ concentrations were calculated from water content of tissue assuming no changes in compartmentation of the Mg 2+ or H20, and 3 mmol Mg 2+ 1-1 in chloroplast stroma of turgid leaves.

Since Mg2÷ was supplied during preincubation of the isolated chloroplasts, the experiment resembled the preincubation conditions that occurred in dehydrating leaves. The concentrations of Mg2÷ necessary to bring about inhibition were similar to the increases in concentration that would be expected simply from water loss by the dehydrated leaf tissue (Table II). It is therefore possible that the concentration of cellular constituents to

244

TABLE

III

Magnesium concentration during preincubation, and p h o s p h o r y l a t i o n a c t i v i t y o f isolated spinach chloroplasts and Ca~÷-ATPase activity of c h l o r o p l a s t c o u p l i n g f a c t o r d u r i n g subsequent assay Ca2 ÷-ATPase activity Mg ~÷ c o n c e n t r a t i o n Photophosphorylating (umol h -~ (mg chloroplast)-I) d u r i n g p r e i n c u b a t i o n activity ( m m o l 1- ~) (~ m o l h - ~ ( m g c h l o r o p l a s t )- 1) Before a c t i v a t i o n A f t e r a c t i v a t i o n 0 5 10

1500 1050 750

21 23 14

21 7 5

Preincubation was carried out in the presence of buffer, and a small aliquot of the preparation was transferred to the assay m e d i u m after 30 rain at r o o m temperature. The assay m e d i u m diluted the M g 2+ concentration to 1 % of the preincubation concentration. Heat activation of coupling factor is necessary to demonstrate high ATPase activities. The M g 2÷ preincubation was therefore done prior to heat activation or after heat activation, followed by assay in the usual way. Details of methods are given in Younis et al. (1979).

which specific photosynthetic reactions are sensitive could have an effect on chloroplast activity as leaf water potentials decline. Translocation of photosynthate continues at low water potentials despite the loss of photosynthetic activity in leaves. Experiments conducted both in laboratory (McPherson and Boyer, 1977) and field (Jurgens et al., 1978) environments showed that leaf water potentials low enough to result in the cessation of dry matter accumulation by the whole plant nevertheless allowed the accumulation of dry matter in developing grain in corn plants. The translocation consisted of stored reserves mobilized primarily from stems. The result was grain production in excess of that contributed by drought-inhibited photosynthesis during the grain filling period. Thus, translocation is less sensitive than photosynthesis to low soil water (McPherson and Boyer, 1977; Jurgens et al., 1978; Sung and Krieg, 1979) and acts to preserve grain production under dry soil conditions. An additional contributor to the losses in p h o t o s y n t h e t i c activity that occur at low water potentials is accelerated leaf senescence. It is c o m m o n l y observed that leaf senescence occurs more rapidly at low than high water potentials and this has the result of removing photosynthetic surface from the crop canopy. In determinate plants, like corn, there oft~en is no regrowth of leaves if water is supplied after flowering takes place and, thus, accelerated leaf senescence can permanently inhibit the potential photosynthetic activity of the crop (Legg et al., 1979). We recently explored the significance of accelerated leaf senescence in corn. Plants were dehydrated during the grain filling period and remained at low soil water potentials until grain maturity. During the treatment, all the leaves senesced. In the controls, however, senescence did not occur

245

until the end of the season. Table IV shows that the controls lost water from the leaves at a rate of 42 mg m -2 s -1 and, after senescence, at 2.8 mg m -2 s -1. The plants growing in dry soil lost water at a rate of 6.9 mg m -2 s -1 and after the leaves senesced, at a rate of 0.7 mg m -2 s -1. Because leaf water potentials had decreased to --1.8 to --2.0 MPa in the plants in dry soil, photosynthesis was negligible (McPherson and Boyer, 1977) and, consequently, grain fill was dependent on stored reserves. At maturity, the grain had accumulated approximately 40 g per plant of dry material from reserves in the vegetative organs {about 25% of control yields). Of this amount, about 8 g per plant was supplied from the leaves and the remainder was supplied by the stems. Thus, the leaves contributed relatively small amounts of dry matter to the grain. TABLE IV Transpiration in maize plants having viable and senescent leaves after water had been withheld during the grain filling period Leaf water potential

Transpiration (rag m-2 s-l)

(MPa)

Viable leaves

Senescent leaves

--0.5 --1.8 t o - - 2 . 0

42 6.9

2.8 0.69

Transpiration was measured in the whole plant before and after leaf senescence and is expressed on a unit leaf area basis. At --0.5 MPa, leaf senescence occurred at the end of the growing season. At --1.8 to --2.0 MPa, leaf senescence was complete 3 weeks after water had been withheld from the soil.

In view of the small a m o u n t of reserves made available to the g a i n and the small a m o u n t of transpiration that was prevented by accelerated senescence of the leaf tissue, it appears that accelerated leaf senescence under dry conditions is an undesirable feature for agriculture. It therefore seems t h a t genetic improvement of crop performance under dry conditions could be based at least in part on genotypes that retain leaf tissue during drought but undergo the usual senescence at maturity. SEVERE DESICCATION

Leaf tissue that does n o t undergo accelerated "senescence must be capable of withstanding significant dehydration. Most higher plants are capable of withstanding desiccation to the air-dry state when they are embryos in seed. After germination, however, metabolic changes cause the seedling to become susceptible to severe dehydration, which usually is lethal. Recently, native species have been found in Africa that can withstand desiccation of leaves to the air-dry state. Hallam and Gaff (1978a, b) and Wellburn and

246

Wellburn (1976) have described the characteristics of these plants at the ultrastructural level. Table V shows that there is loss of integrity or damage in virtually all organelles except the nucleus of leaf cells of these plants during severe desiccation. In contrast, the nucleus is damaged and, indeed, disappears during severe leaf desiccation in sunflower, which is not desiccation tolerant. These results suggest that the maintenance of many types of cellular organelles is not necessary for severe desiccation, but that maintenance of the nucleus is essential. This implies that survival of severe desiccation requires the cell to repair and/or reconstruct organelles outside the nucleus. Thus, the metabolic basis of cell tolerance to severe desiccation may reside in mechanisms that protect nuclear integrity. TABLE V A l t e r e d u l t r a s t r u c t u r e in cells o f leaves o f higher plants in the air-dry state; X e r o p h y t a , Talbotia, and M y r o t h a m n u s are d e s i c c a t i o n t o l e r a n t , H e l i a n t h u s is d e s i c c a t i o n sensitive Structure

Xerophyta villosa a

Talbotia elegans b

Myrothamnus flabellifolia c

Helianthus annuus d

Nucleus Chloroplast envelope Chloroplast thylakoids Mitochondria Golgi b o d i e s Tonoplast Plasmalemma

0 + ++ ++ -. . --

-++ + ++ ++

0 ++ + 0 + ++ --

++ ++ + ++ ++ ++ ++

.

. ++

0, shape change; +, m o d e r a t e change in integrity; ++, major change in i n t e g r i t y ; - - , n o observation. aHallam and G a f f ( 1 9 7 8 b ) ; b H a l l a m and G a f f (1978a); CWellburn a n d Wellburn (1976); d F e l l o w s a n d B o y e r (1976). CONCLUSIONS

Several opportunities for improving the drought tolerance of plants are suggested by these results. First, the accelerated senescence of leaf tissue so frequently observed under dry conditions in crop plants could probably be selected against without sacrificing much yield. If accelerated senescence did not occur, photosynthesis could resume when water was resupplied to the crop. Second, the survival of leaf tissue in the air dry state in certain species indicates that drought tolerance can be attained by plant~, during vegetative growth. In fact, the embryos of most seed plants can withstand dehydration to the air dry state, but this ability is only rarely expressed at later stages of growth. The significance of nuclear stability in desiccation tolerant cells remains to be assessed, but the stability implies that there are cellular properties that can protect organelles during severe desiccation. Third, with evidence accumulating that osmotic adjustment

247 p e r m i t s g r o w t h u n d e r d r y c o n d i t i o n s , and t h a t genetic variability exists in this trait, t h e i m p r o v e m e n t o f o s m o t i c a d j u s t m e n t in c r o p species m a y e n h a n c e p r o d u c t i o n u n d e r d r y c o n d i t i o n s . T h e beneficial effects o f o s m o t i c a d j u s t m e n t are likely t o be m o d e s t , because rapid g r o w t h is o f t e n sacrificed t o allow solute t o a c c u m u l a t e in t h e growing tissue. H o w e v e r , even a m o d e s t gain m i g h t translate into significant e c o n o m i c benefits.

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