Brassinosteroids and Root Development - ACS Publications

Chapter 20 Brassinosteroids and

Root Development

Downloaded by NORTH CAROLINA STATE UNIV on January 7, 2013 | http://pubs.acs.org Publication Date: November 4, 1991 | doi: 10.1021/bk-1991-0474.ch020

J. G. Roddick and M . Guan

1

Department of Biological Sciences, University of Exeter, Exeter, United Kingdom

Relatively little i s known about the e f f e c t s of brassinosteroids (BS) on root growth and development and nothing at all concerning t h e i r occurrence i n roots. Results are v a r i a ble, but BS mainly i n h i b i t root development i n cuttings and seedlings, and at low concentrations (10 μM). Simultaneous, but variable, e f f e c t s may occur on the shoot, but cultured excised root studies suggest that root i n h i b i t i o n is a direct e f f e c t . Promotion of root growth i s reported for cuttings, normal and stressed seedlings, and segments, but some of these conclusions are open to question. In segments, BS show opposite e f f e c t s to IAA on proton extrusion, Δ pH and membrane p o t e n t i a l . BS appear to be transportable i n cultured roots and t h i s system may be p a r t i c u l a r l y useful for further studies of BS e f f e c t s on roots. Following the discovery of 'brassin' i n 1970 (1), the i s o l a t i o n and chemical characterization of the active ingredient brassinolide (BR) i n 1979 (2) and the subsequent i d e n t i f i c a t i o n of a number of other naturally-occurring brassinosteroids (BS) (see 3), considerable research e f f o r t has been directed towards determining the taxonomic and morphological d i s t r i b u t i o n and b i o l o g i c a l a c t i v i t y of these compounds i n plants. Studies of the occurrence of BS i n the plant kingdom have shown them to be present i n v i r t u a l l y a l l higher plants examined so far from a wide range of families (3,4), and also i n the u n i c e l l u l a r green alga Hydrodictyon reticulatum (5). Considering t h i s trend, i t i s not beyond the bounds of p o s s i b i l i t y that BS may prove to be universal constituents of (at least higher) plants. Within the plant i t s e l f , BS have been detected i n almost every organ, a l b e i t at variable (but low) l e v e l s , as summarized i n Table I. 1

Current address: Diagnostic Biotechnology (Pte) Ltd., 65 Science Park Drive, Singapore Science Park, Singapore 0511

0097-6156/91/0474-0231$06.00/0 © 1991 American Chemical Society In Brassinosteroids; Cutler, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Table I .

BRASSINOSTEROIDS: CHEMISTRY, BIOACTIVITY, AND APPLICATIONS O c c u r r e n c e and

L e v e l s o f BS i n H i g h e r P l a n t T i s s u e s / O r g a n s

Tissue/Organ

Cone.
Pollen Seeds Fruits Flower buds Shoots Leaves Galls Cultured crown-gall

5 0.2 Trace 3 0.008 0.0005 0.01 30

tissue

-

-

190 5 1 15 27 30 26 40

Ref

•

6 7 - 9 10 - 12 9, 13 12 - 14 10, 13, 15 10, 12 16

Notable by i t s absence from t h i s l i s t i s any reference to roots or other subterranean structures. The importance of roots as absorpt i v e , anchorage and storage organs needs no elaboration here and yet a l l analyses of plant material for BS so far conducted have been confined to a e r i a l tissues. Amazingly, t h i s oversight has persisted for more than a decade. Arguably, the most eminent b i o l o g i c a l e f f e c t of BS i s t h e i r a b i l i t y to enhance shoot elongation and growth i n a variety of systems: excised segments, cuttings, seedlings and plants (17,18) . Other important developmental e f f e c t s of BS include stimulation of pollen tube growth (19), s e n s i t i z i n g tissues to gravistimulation (20), induction of epinasty (21) and accelerating flower production and a l t e r i n g the flower sex r a t i o (22) . The p o t e n t i a l importance of such e f f e c t s i s apparent from the fact that application of BS to the shoots of a number of crop plants can produce greater or e a r l i e r crop y i e l d s (4,17,18,23). Once again, however, roots have been sadly neglected i n studies into the b i o l o g i c a l a c t i v i t y of BS, although not t o t a l l y so. A r e l a t i v e l y small number of published reports exist i n t h i s f i e l d which c o l l e c t i v e l y indicate that BS are capable of a l t e r i n g developmental processes i n roots at low concentrations. Unfortunately, some of these reports are based on i n c i d e n t a l observations and/or are not s u f f i c i e n t l y quantified. The consequent uncertainties are further compounded by an element of v a r i a b i l i t y between some reports. As a r e s u l t , no clear picture has yet emerged of the developmental significance of BS i n roots and we are l e f t with a very unbalanced knowledge of the d i s t r i b u t i o n and possible importance of BS i n the plant body as a whole. At t h i s stage therefore, there exists a need to bring together the various scattered reports of BS e f f e c t s on root growth and development i n order to assess c r i t i c a l l y the present status of these compounds i n t h i s important organ and to i d e n t i f y directions i n which future work i n t h i s f i e l d might proceed. Despite the small number of studies on the influence of BS on roots, a l l of the most important experimental root systems, that i s , adventitious cutting roots, i n t a c t seedling/plant roots, excised root segments and excised cultured roots have already been employed.

In Brassinosteroids; Cutler, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Information r e l a t i n g to these d i f f e r e n t systems i s reviewed i n the four major sections of t h i s a r t i c l e .


Adventitious roots Probably the e a r l i e s t investigation of the e f f e c t s of BS on adventitious root formation i s described i n a two-part report published i n 1981 by Yopp et a l . (24) and Mandava et a l . (25). The former study used mung bean (Phaseolus aureus cv. Berken) cuttings i n which adventitious root development normally responds well to auxin. While the number of adventitious roots emerging from the hypocotyl was unaffected by 0.1 BR, i t was more than halved by 1 \xM BR and t o t a l l y suppressed by 10 BR. The lengths of adventitious roots were not recorded, but shoot length was stimulated by increasing concentrations of BR. This i n h i b i t o r y e f f e c t of BR on rooting i s of considerable i n t e r e s t , contrasting as i t does with the promotory e f f e c t of IAA i n t h i s system (24), whereas i n azuki bean (Vigna angularis cv. Takara) e p i c o t y l s , BR and IAA show s i m i l a r growth-enhancing effects (24,26). These findings have been interpreted as demonstrating that BR i n h i b i t s root emergence but not root i n i t i a l format i o n (4); however, t h i s conclusion i s probably not warranted i n the absence of h i s t o l o g i c a l examination of the hypocotyl. Dihydroc o n i f e r y l alcohol and chlorogenic acid, which synergize some gibbere l l i n and auxin responses respectively, had no e f f e c t on BR i n h i b i tion of rooting i n mung bean (P. aureus) hypocotyls (27). The second part of the study (25) e s s e n t i a l l y confirmed i n h i b i tion of adventitious rooting by BR i n other species v i z . bean and cucumber, although the observations were i n c i d e n t a l and the e f f e c t s not quantified. In e t i o l a t e d bean (Phaseolus vulgaris cv. Executive) hypocotyls o r i g i n a l l y used to study hook opening, treatment with 10 BR suppressed adventitious root development. E t i o l a t e d cucumber (Cucumis sativus cv. Long Green) hypocotyls lacking cotyledons normally used for elongation experiments also f a i l e d to produce adventitious roots when treated with 10 \xM BR. In cotyledon-bearing hypocotyls, roots never formed at the base i n the presence of BR, although some did appear approximately 1 cm from the basal end. Although g i b b e r e l l i n s strongly i n h i b i t adventitious root development i n a number of plant systems (28), the authors (25) caution that t h i s BR e f f e c t cannot necessarily be regarded as ' g i b b e r e l l i n - l i k e ' as cytokinins are also capable of i n t e r f e r i n g with rooting (29). Recent work of a more quantitative nature (30,31) provides further support for an i n h i b i t o r y e f f e c t of BS on adventitious root formation. In hypocotyl and e p i c o t y l cuttings from mung bean (P. aureus), both the number of roots produced and the mean root length were markedly reduced by 24-epibrassinolide (epiBR) (30). Rooting was s i g n i f i c a n t l y and consistently impaired by 0.1 p BS with maximal e f f e c t s at 1.0 \iM. These e f f e c t s on root number and elongation compounded to produce s i g n i f i c a n t reductions (approx 50%) in t o t a l root length for each type of cutting at 0.01 pM (Table I I ) . M


234

BRASSINOSTEROIDS: CHEMISTRY, BIOACTIVITY, AND APPLICATIONS

Table I I . A p p r o x . C o n c e n t r a t i o n (yM) o f EpiBR p r o d u c i n g 50% I n h i b i t i o n o f A d v e n t i t i o u s Root Growth i n C u t t i n g s Growth parameter Species

(Type) Number of Roots


Mung Bean (hypocotyl) Mung bean (epicotyl) Tomato SOURCE:

0.01 0.05 0.1

Adapted from r e f . 30,

Mean Root Length 0.1 0.07 0.7

Total Root Length 0.01 0.01 0.04

31.

In tomato (Lycopersicon esculentum cv. Best of A l l ) cuttings, epiBR again caused a general i n h i b i t i o n of rooting, although individual parameters responded somewhat d i f f e r e n t l y from mung bean (31). With increasing concentrations up to 1 pM, the number of roots produced declined, but at 10 \iM there was an upturn i n the graph and cuttings produced a greater number of roots (roughly equivalent to that i n controls) higher up on the hypocotyl. However, mean root length progressively decreased with increasing epiBR concentration with the result that t o t a l root length per cutting showed an essent i a l l y inverse r e l a t i o n s h i p with BS strength. The s e n s i t i v i t y of adventitious roots of tomato to epiBR appeared less than that of mung bean roots (Table I I ) . In both mung bean and tomato cuttings, increasing levels of epiBR not only progressively reduced rooting but simultaneously stimulated shoot elongation, e s p e c i a l l y e p i c o t y l elongation. Such d i f f e r e n t i a l growth e f f e c t s are i n keeping with s i m i l a r e a r l i e r observations i n mung bean (24) and cucumber (25) . The former authors (24) concluded that t h i s e f f e c t was not a r e s u l t of prefere n t i a l nutrient diversion to a e r i a l organs. The reasons for i t s t i l l remain unclear, but i t i s tempting to speculate that root development might be more sensitive to BS than shoot tissues such that levels of BS promotory to shoots might be i n h i b i t o r y to roots, a s i t u a t i o n which i s known for auxins. Nevertheless, these data do not rule out the p o s s i b i l i t y that e f f e c t s on root development may be somehow mediated v i a e f f e c t s on shoot tissues (but see below). In contrast to the above, Vorbrodt et a l . (32) reported that the development of roots from cloned, excised shoots of Matricaria chamomilla cultured on a g a r - s o l i d i f i e d medium was increased by between 2-6 times by treatment with 0.001-10 ppm (2nM-20pM) (22S, 23S) homobrassinolide (homoBR) either incorporated into the agar medium or applied by dipping into a powder or solution. Low concentrations of epiBR have also been found to enhance rooting i n excised soybean (Glycine max) hypocotyls under certain conditions (33) . Hypocotyls from seedlings raised i n continuous l i g h t or darkness were not responsive to the BS, whereas long-day-grown (16h) tissues responded maximally i n terms of both root number and length to 0.0001 ppm (0.2 nM) for 8h. The e f f e c t of 0.001 ppm (2 nM) for 4h was also


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s i g n i f i c a n t l y greater than the control. Where roots increased i n number, they were present over a greater length of the hypocotyl which the authors suggest might have resulted from a more extensive d i s t r i b u t i o n of epiBR i n the hypocotyl. No i n h i b i t o r y e f f e c t s of epiBR were apparent even at the highest concentration (200 nM) .


Seedling roots The response of seedling roots to exogenous BR also figures i n the early communication of Yopp et a l . ( 2 4 ) . In dark-grown cress (Lepidium sativum cv. Curley) seedlings, root growth was i n h i b i t e d by IAA at 0.1 pM or greater, but l i t t l e affected either way by up to 10 \iM BR. Possibly as a result of t h i s 'neutral' e f f e c t , no major quantitative study of the effects of BS on seedling roots appeared for a number of years. A few sporadic mentions of root e f f e c t s were reported but, because of t h e i r q u a l i t a t i v e nature, these made no s i g n i f i c a n t contribution to the knowledge base. For example, spraying a 1 (iM solution of an u n i d e n t i f i e d BS on to hydroponically-grown wheat (Triticum aestivum cv. K o l i b r i ) seedlings promoted photosynthesis, soluble reducing sugars, and growth of the shoot and root, but none of the data presented applied to the root e f f e c t (34). In his recent review, Mandava (4) c i t e s unpublished work by Gregory claiming that BR "induces both root and leaf growth i n lettuce" (Lactuca sativa), but again no supporting experimental data are available. Seedlings of radish (Raphanus sativus cv. Tokinashi) and tomato (L. esculentum cv. Giant Cherry) with t h e i r roots immersed i n BS solutions for short periods have been used i n bioassays, but only shoot growth was measured ( 3 5 ) . In a more quantitative study, Luo et a l . (36) also noted enhancement of photosynthesis i n wheat (T. aestivum) by BR as well as of primary root formation i n seedlings. Root (and shoot) elongation was not stimulated by BR but, i f anything, s l i g h t l y i n h i b i t e d by higher concentrations. Promotion of shoot growth but i n h i b i t i o n of root growth was recorded i n radish (R. sativus) seedlings sprayed with 0.1 ppm (0.2 pM) BR with the e f f e c t being less pronounced under long days than short days ( 3 7 ) . EpiBR was likewise found to promote shoot growth but reduce root growth (as measured by dry weight) i n hydroponically-treated tomato seedlings ( 3 1 ) . Seedling roots did not appear as sensitive as cutting roots being hardly affected by 0.01 pM epiBR, although root weight was reduced by approximately 40% by 1 JJM. This d i f f e r e n t i a l s e n s i t i v i t y may be a result of d i f f e r e n t rates of uptake by, and transport within, intact and excised organs. More recent work by one of us (JGR), currently being prepared for publication, has confirmed the a b i l i t y of low levels of epiBR to i n h i b i t root development i n hydroponically-grown seedlings of other species e.g. mung bean (P. aureus), wheat (T. aestivum cv. S i r Galahad) and maize (Zea mays cv. Santos) (Table I I I ) . In the f i r s t two species, main root number and extension growth were unaffected by the BS, but l a t e r a l root number and


236


Table I I I .

Approx. Concentration (pM) of EpiBR producing I n h i b i t i o n of Seedling Root Growth

Species

Growth parameter Main Root Number

Wheat Mung Bean Maize Downloaded by NORTH CAROLINA STATE UNIV on January 7, 2013 | http://pubs.acs.org Publication Date: November 4, 1991 | doi: 10.1021/bk-1991-0474.ch020

50%

N.I. N.I. 10

Main Root Length N.I. N.l/ >10?

Lateral Root Number

Total Lateral Root Length 0.01 0.1

1 10 N.I.

No i n h i b i t i o n with up to 10 pM Inhibition < 50% at maximum tested concentration

7

(10 \iM) .

elongation were depressed, although the magnitude of the e f f e c t s varied. Wheat also showed considerable morphological d i s t o r t i o n of the root. Maize, on the other hand, d i f f e r e d i n i t s response and s e n s i t i v i t y i n that the growth of the main root was affected as was l a t e r a l length (but not l a t e r a l number), but some e f f e c t s were only observed at, or approaching, the highest concentration (10 pM). EpiBR had no e f f e c t on shoot growth i n wheat or maize but i n the mung bean seedlings, concentrations of 0.1 pM or greater consistently i n h i b i t e d e p i c o t y l extension growth. This contrasts with the promotion of elongation i n mung bean cuttings (24, 30) and i n tomato seedlings (31), but the reasons for t h i s are not yet known. Of considerable i n t e r e s t as well as potential importance, i s the very recent claim (38) that i n h i b i t o r y e f f e c t s of s a l t stress (500 ppm NaCl) on root growth i n r i c e (Oryza sativa cv. Nihonbare) seedlings could be e s s e n t i a l l y n u l l i f i e d by pre-soaking seeds i n _ homoBR solutions for 48h. A l l tested concentrations from 10"" - 10 ppm (2 pM - 200 nM) were equally e f f e c t i v e . Unfortunately, data from control seedlings raised i n the absence of NaCl are not i n cluded and so the extent of recovery of root growth attributed to homoBR cannot be properly gauged. The same authors (38) also studied the e f f e c t of epiBR on root growth i n r i c e (O. sativa cv. Aepung) seedlings the seeds of which had previously been stored at 5°C for d i f f e r e n t lengths of time v i z . 3 or 5 years. In the l a t t e r , root elongation was approximately 25% of that i n the former, but could be p a r t i a l l y restored (to about 55-65%) by the presence of low concentrations of epiBR ( 1 0 ~ - 10~ ppm, 0.002 fM - 2 pM). Enhancement of root growth by epiBR i n the 3-year-old treatment was much less and possibly i n s i g n i f i c a n t . It would have been useful to know how growth and responses to epiBR i n 3- and 5-year-old treatments compared with those i n fresh seeds, but t h i s treatment was not included. Incidentally, an e f f e c t of epiBR at 1 0 ~ ppm (0.002 fM) must rank as one of ( i f not the) lowest active concentrations of BS recorded. 1

6

12

12


6

20. RODDICK & GUAN

237

Root Development


Root segments The fact that BS and auxins exert quite d i f f e r e n t e f f e c t s on root development i n cuttings and seedlings (24) was very much i n the minds of Bonetti, Romani, Cerana and co-workers when they embarked on a programme of investigations into the e f f e c t s and mechanisms of BS on root segments. As background to t h i s work also was t h e i r e a r l i e r finding (26) that epiBR (like IAA) increases a c i d i f i c a t i o n of the walls of azuki bean e p i c o t y l c e l l s probably through a stimulation of electrogenic proton extrusion. Working with 5 mm a p i c a l and sub-apical segments of maize (Z. mays cv. DeKalb XL 640) roots, treatment with 3 pM epiBR i n creased extension growth by more than twice that i n controls (39, 40). This e f f e c t was accompanied by a reduction i n medium pH, the magnitude of which was related to [K ] i n the medium. This was interpreted as r e s u l t i n g from an antiport exchange of K and H v i a an electrogenic proton pump. Further evidence for H extrusion was a hyperpolarization of the membrane p o t e n t i a l and i n h i b i t i o n of the BS e f f e c t s by the protonophore (_-trifluoromethoxy)-carbonylcyanidephenylhydrazone (FCCP). These findings (summarized i n Table IV) further demonstrated that BR and IAA e f f e c t s on roots also d i f f e r at the cellular/biochemical l e v e l , the l a t t e r causing i n h i b i t i o n of root segment elongation, increase i n pH and depolarization of the membrane p o t e n t i a l (39, 40) . +

+

+

+

Table IV. E f f e c t of EpiBR on Growth o f Maize Root Segments, Medium pH, Proton Extrusion and Membrane P o t e n t i a l a a Treatment Mean % Increase A pH Proton Extrusion Membrane i n Length (pmol/g FW) P o t e n t i a l (mV) a

Control 1 pM BR 3 pM BR 10 \iM BR

5.,3 12..8

13

-0.53 -0.92 -0.86 -0.84

2,.04 3..27 3..15 3,.11

-105 -125 -124

Sub-apical 5 mm segments incubated i n Na -MES buffer for 4h. SOURCE: Adapted from r e f . 39. Apical 2.5 cm segments i n Na -MES buffer a f t e r 30 min. SOURCE: Adapted from r e f . 40.

a

+

b

A comparative study (41) of the e f f e c t s of epiBR, twelve BS analogues and three s t e r o l s (stigmasterol, ergosterol and cholesterol) on growth and proton extrusion i n sub-apical maize (Z. mays cv. DeKalb XL 72) root segments revealed f i v e analogues to be v i r t u a l l y as active as epiBR on root growth, the other seven of reduced a c t i v i t y and the s t e r o l s i n a c t i v e . These data confirmed that the s t r u c t u r a l requirements for BS a c t i v i t y i n roots were the same as those for a c t i v i t y i n shoots (42). Of i n t e r e s t however was that a l l the BS tested, whether of high or low growth a c t i v i t y , as well as the


238


growth-inactive sterols altered the external pH to the same extent. Cholesterol also proved to be as active as epiBR i n stimulating K uptake into the segments and dark CO2 f i x a t i o n . Further investigations (4_3) confirmed the importance of K (but not Na or Cl~) i n epiBR and cholesterol enhancement of H extrusion and related t h i s to an increased rate of K i n f l u x (as measured by Rb) and proton pumping. In addition, growth stimulation i n root segments by BS was shown not to be a result of an increase i n the concentration of osmotically-active c e l l solutes. Although possible reasons were offered for the finding that sterols a l t e r pH and H extrusion but not growth (43), t h i s d i s crepancy casts some doubt on the involvement of a c i d i f i c a t i o n processes i n BS-induced growth. These doubts may also be compounded by closer examination of the data f o r growth i n root segments which are expressed on a percentage basis. The minimum and maximum % i n creases i n growth i n control segments were 4.9% and 9% respectively while the corresponding values f o r the 3 pM epiBR treatments were 13.8% and 16% (40, 41). In 5 mm segments, these values represent growth increments of only 0.25 mm and 0.45 mm i n controls, and 0.69 mm and 0.8 mm i n BS treatments. No d e t a i l s were given of how extension growth i n root segments was measured and although the values may be s t a t i s t i c a l l y acceptable, the smallness of the changes renders the b i o l o g i c a l significance of these data uncertain. In similar experiments with a p i c a l and sub-apical root segments from wheat (T. aestivum cv. S i r Galahad), mung bean (P. aureus) and maize (Z. mays cv. Santos) we were unable to detect any s i g n i f i c a n t BSinduced changes i n l i n e a r dimensions (Table V). Although we did not subject segments to the elaborate pretreatments i n CaSC>4 solutions used i n the above work, we nevertheless obtained elongation equivalent to, or i n excess of, that detailed above. Despite these +

+

+

+

+

86


+

Table V.

E f f e c t of EpiBR on Elongation of Excised Root Segments

Species (Type of segment ) 13

Wheat (A) Wheat (SA) Maize (SA) Mung Bean (SA)

Initial Length (mm)

0

5.10 5.10 5.02 4.85

6.44 5.93 5.44 5.41

F i n a l Length (mm) BR Cone. (pM) 0.1 0..01 1 6..47 5..78 5..59 5..34

6.54 5.50 5.52 5.31

6.73 5.52 5.52 5.28

3

10 6.43 5.47 5.39 5.22

a Segments incubated i n the dark at 25°C f o r 5h, except wheat (7h). Lengths measured using a photographic enlarger (x 5). Values are means of 10 r e p l i c a t e s . S.E. range was ± 0.06 - ± 0.15. A = a p i c a l , SA = subapical from 3 mm below apex. b

reservations, root segments may o f f e r a convenient method f o r studying the uptake, transport and possible binding c h a r a c t e r i s t i c s of BS. This was the rationale underlying work by A l l e v i et a l . (44) i n


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which the movement of deuterated epiBR into maize (Z_ mays cv. DeKalb XL 72) root segments was followed by multiple selected ion monitoring. The BS quickly accumulated i n root tissues by nonenergy-dependent processes possibly involving adsorption to c e l l membranes. However, a comparison of the uptake and release patterns of epiBR i n l i v i n g and frozen tissues revealed that i n the former there i s a greater element of i r r e v e r s i b l e binding of BS. How t h i s i s achieved s t i l l awaits c l a r i f i c a t i o n .


Cultured excised roots Convenient as root segments may be for some work, t h e i r usefulness i s diminished by the fact that they represent only a f r a c t i o n of the root system thus making i t d i f f i c u l t to extrapolate to the whole root. Adventitious and seedling roots do not present such problems, but t h e i r physical association with shoot tissues can make for d i f f i c u l t i e s i n determining whether roots respond d i r e c t l y to BS or i n d i r e c t l y v i a primary e f f e c t s on the shoot. One approach which may circumvent both these methodological problems i s to grow excised roots i n i s o l a t i o n i n a s t e r i l e l i q u i d culture medium. Using tomato roots (L. esculentum cv. Best of A l l ) grown i n t h i s way, we have established that roots are d i r e c t l y i n fluenced by BS (31). Concentrations of epiBR between 1 pM and 1 nM did not a f f e c t root growth, but 10 nM s i g n i f i c a n t l y i n h i b i t e d elongation of the main root axis and the number of l a t e r a l roots produced. In both these parameters and also i n t o t a l extension of l a t e r a l s , growth was t o t a l l y suppressed by 1 pM epiBr. These findings (Figure 1) suggest that root i n h i b i t i o n by BS i n tomato seedlings and cuttings (31), mung bean cuttings (24) and cucumber cuttings (25) where e f f e c t s on shoot tissues simultaneously occurred i s probably mainly due to a d i r e c t e f f e c t on the root system, although i n t e r active e f f e c t s deriving from the shoot cannot be t o t a l l y ruled out. Excised tomato roots grown i n s t e r i l e culture appeared to be more sensitive to epiBR than t h e i r intact counterparts i n seedlings, which might be explained i n a number of ways. For example, greater access of BS into excised roots v i a the cut surface, lack of microb i a l degradation/transformation of test compounds i n s t e r i l e conditions, altered hormonal/nutrient status of cultured roots as a result of excision, etc. In t h i s respect, the generally greater responsiveness to auxin of excised plant parts compared with i n t a c t structures should be borne i n mind. Also, because cultured excised roots responded i n e s s e n t i a l l y the same way as adventitious and seedling roots, they could be useful i n o f f e r i n g a more sensitive and controlled system for investigating BS e f f e c t s on roots. To date, most experimental work demonstrating e f f e c t s of BS on root development has involved exogenous presentation of the t e s t steroids to the root (or rooting) medium. Without doubt, t h i s bears no resemblance to the i n vivo s i t u a t i o n where roots would respond (if at a l l ) to endogenous BS either synthesized within the root or transported from the shoot. As a f i r s t step towards assessing the



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responsiveness of roots to endogenously-supplied BS, we have presented epiBR to the basal or a p i c a l ends of cultured excised tomato roots and measured subsequent growth i n the corresponding d i s t a l regions of the root. This work i s s t i l l of a preliminary nature, but early r e s u l t s indicate that main axis extension growth and l a t e r a l root development can be markedly i n h i b i t e d by BS applied at some other part of the root (Figure 2). With external transport along the root e s s e n t i a l l y prevented by a vaseline b a r r i e r , our data suggest f i r s t - that roots are able to respond to BS supplied endogenously, and second - that BS are transportable both acropetally and b a s i p e t a l l y i n root systems. It i s not yet known i f epiBR i s transported i n t h i s or a derivatized (e.g. glucosylated) form. Whether BS are a c t u a l l y transported within roots or between roots and shoots (or within shoots) i n intact plants has yet to be established. BS are obviously and e f f e c t i v e l y transported into cuttings v i a the transpiration stream, but t h i s i s not evidence of transport i n vivo. Also e f f e c t s on root (and shoot) growth following a seed soak i n BS solutions (38) or spray application of BS to the shoot (34) cannot be regarded as unequivocal proof of BS transport to the roots or a response by t h i s organ to endogenouslysupplied BS. Obviously, much remains to be done i n t h i s p o t e n t i a l l y important area of BS physiology and, once again, cultured excised roots might o f f e r a valuable, complementary approach to some of the more intractable problems. Discussion and

conclusions

Because of the r e l a t i v e l y small amount of work done i n t h i s f i e l d and the v a r i a b i l i t y which exists between some reports, i t i s d i f f i cult to a r r i v e at a consensus regarding the e f f e c t s of BS on roots. Nevertheless, there does appear to be a certain weight of evidence that low concentrations of exogenously- (and possibly endogenously-) supplied BS can cause serious impairment of root development. This is a p a r t i c u l a r l y i n t e r e s t i n g phenomenon because i t stands i n complete contrast to the powerful promotory e f f e c t s of BS on shoot growth and raises i n t e r e s t i n g questions not only about the mechanism of action of BS (which s t i l l remains unresolved), but also about whether the mechanism (or consequences) might d i f f e r i n d i f f e r e n t parts of the plant. Of possible relevance i n t h i s respect are the observations that BS and auxin appear to produce opposite e f f e c t s i n root systems whereas they usually act i n a s i m i l a r (but not i d e n t i c a l ) way on the shoot and commonly show important i n t e r a c t i v e e f f e c t s . The most s t r i k i n g l y d i f f e r e n t e f f e c t s of these two compounds i n roots r e l a t e to adventitious root formation i n cuttings which i s i n h i b i t e d by BS (24, 30, 31) and promoted by auxin (24), and proton secretion and membrane p o t e n t i a l i n root segments which are enhanced and hyperpolarized respectively by BS and i n h i b i t e d and depolarized respect i v e l y by auxin (39, 40). These data could suggest either that BS act independently of auxin i n roots or that they antagonize



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RODDICK &GUAN

Root Development

EpiBR cone. C juM)

Figure 1. E f f e c t of epiBR on growth of cultured excised tomato roots after 7 days. Inocula were 10 mm with no l a t e r a l s . Points represent the mean of 10 replicates and v e r t i c a l bars the S.E. (Adapted from r e f . 31).

Figure 2. Tracings of t y p i c a l cultured excised tomato roots treated at the a p i c a l or basal end with 10 pM epiBR for 7 days. The two-well treatment/culture vessel i s shown inset. In Brassinosteroids; Cutler, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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endogenous auxin (4, 39). On the other hand, BS are capable of stimulating ethylene synthesis i n shoot tissues and can synergize with auxin i n t h i s respect (45-47). It i s also well known that root growth can be suppressed by ethylene (48, 49) and thus the p o s s i b i l i t y that ethylene release by root tissue may be highly sensitive to BS stimulation might be worthy of exploration. _ Testing a wide range of epiBR concentrations (10 - 10 M) on cultured excised tomato roots (31) did not identify a clear optimum concentration suggesting that the i n h i b i t o r y e f f e c t s on roots are possibly not due to greater s e n s i t i v i t y of root c e l l s than shoot c e l l s to BS. On the other hand, Zhao (Shanghai I n s t i t u t e of Plant Physiology, personal communication, 1990) found lower concentrations of BS (e.g. 0.1 yM) stimulated root growth i n wheat (T. aestivum) while higher concentrations were i n h i b i t o r y , as did also Luo et a l . (36). Also, the BS dose-response curve for root growth i n r i c e seedlings from 5 year old seeds (38) could be taken as some evidence that BS produce an optimal e f f e c t at very low concentrations. Whether these r e s u l t s r e f l e c t species s p e c i f i c responses i s not yet known. In order to r a t i o n a l i z e some of the c o n f l i c t i n g reports of BS on roots, further careful attention needs to be given to the mode, l e v e l and time of exposure to BS, a l l of which are i n t e r - r e l a t e d i n a complex manner through events such as uptake, transport and compartmentation. Exogenous application i s informative, but highly a r t i f i c i a l , and i t would be preferable to adopt a l t e r n a t i v e systems in which BS a r r i v e at target c e l l s by an endogenous route. The use of excised root cultures could prove p a r t i c u l a r l y valuable here although such a system i s highly r e l i a n t on another major unknown r e l a t i n g to BS, v i z . transport within (or between) organs. The i n creasing a v a i l a b i l i t y of r a d i o i s o t o p i c a l l y - l a b e l l e d BS w i l l hopef u l l y help c l a r i f y t h i s s i t u a t i o n soon. Ideally, the l e v e l of, and exposure times to, BS should be kept to a minimum, but t h i s may not be r e a l i s t i c i f problems e x i s t i n the uptake or transportation of these compounds. In fact the apparent lack of e f f e c t of BS on cress seedling roots i n an early study (24) could possibly be due to the treatment time being only 17h. While i n h i b i t o r y e f f e c t s of BS on rooting and root growth can be c r i t i c i z e d on the basis of the lengths of time of exposure (e.g. up to several days), i t should be noted that s i g n i f i c a n t enhancement of growth i n some shoot tissues requires s i m i l a r treatment times (50). At the same time, claims that BS stimulate root growth or development cannot be t o t a l l y dismissed, although some reservations must attach to c e r t a i n reports because of the smallness of the effects (39-41) or the absence of controls (38). Even so, the p o s s i b i l i t y that BS may exert quite d i f f e r e n t e f f e c t s on roots exposed to stress conditions (see 38) compared with non-stressed organs could have p a r t i c u l a r l y important p r a c t i c a l implications (cf 4). It seems u n l i k e l y that the enhancement of rooting i n soybean hypocotyls (33) but i n h i b i t i o n of t h i s process i n mung bean hypocotyls (24, 30) can be attributed to the species difference, although



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too few species have been studied so f a r to rule t h i s out as an important determinant of response. More l i k e l y i n t h i s case (but as yet inexplicable), i s that the BS e f f e c t was somehow conditioned by the l i g h t i n g regime, since seedlings raised i n long (16h) days produced responsive hypocotyls while those raised i n constant l i g h t or darkness d i d not. That root tissues are highly (and for reasons not yet understood, variably) responsive to applied BS i s now beyond question. Although much remains to be explained regarding the nature, s p e c i f i c i t y and mechanisms of these e f f e c t s , of probably greater importance at t h i s stage i s determining whether or not BS are a c t u a l l y present i n root tissues and, i f so, whether t h i s i s due to biosynthesis within the root or transport from elsewhere. U n t i l such information becomes available, i t w i l l not be possible to place most of the findings described here into any meaningful p h y s i o l o g i c a l context. Acknowledgments We thank Professor N. Ikekawa for g i f t s of epiBR and Anna Rijnenberg for technical assistance. Literature cited

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R E C E I V E D May 13, 1991


Brassinosteroids and Root Development - ACS Publications

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