J Plant Growth Regul (2003) 22:276–288

DOI: 10.1007/s00344-003-0062-3

Physiological Actions of
Brassinosteroids: An Update
Jenneth M. Sasse
School of Resource Management, Institute of Land and Food Resources, The University of Melbourne,
Parkville, Victoria, Australia 3010

ABSTRACT
In general, this overview covers literature from include phenotypic variability, reproductive physi-
1999 until early 2003. Topics covered include as- ology, senescence, branching, and apical domi-
pects of the biosynthesis and transport of brassino- nance, whereas topics in which possible roles for
steroids, their effects on cell division, expansion, BRs are relatively unexplored include lignification,
and differentiation, and their effects on whole phototropism, photoperiodism, and endogenous
plants, including source-sink relations and other rhythms.
endogenous interactions. Some interactions with
environmental signals are discussed, as well as re- Key words: Brassinolide; 24-Epibrassinolide;
sults that may promise applications in future. Topics Castasterone; Cross-talk; 28-Homobrassinolide;
that warrant further investigation of the roles of BRs Plant development; Plant hormones

INTRODUCTION defense are receiving increasing attention. More

details of the networked pathways for the biosyn-
Reviews on brassinosteroids (BRs) that have ap- thesis, conjugation, and metabolism of BRs are
peared since that of Clouse and Sasse (1998) include emerging, and exploration of the value of selected
two books (Sakurai and others 1999; Khripach and or structurally modified BRs for agricultural use
others 1999) and articles which focus on particular continues. Reports also extend to their effects on
aspects of their structures and biology (for example, animal and human physiology. The present over-
Li and Chory 1999; Khripach and others 2000; view updates a chapter on the physiological actions
Schumacher and Chory 2000; Müssig and Altmann of BRs (Sasse 1999).
2001; Friedrichsen and Chory 2001: Bishop and
Yokota 2001; Clouse 2001, 2002ab; Zullo and Adam
2002; Bajguz and Tretyn 2003; Zullo and others OCCURRENCE, SYNTHESIS, AND
2003; Fujioka and Yokota 2003). There has been TRANSPORT OF BRS
impressive progress in our understanding of BR-
induced signaling as well as BR-induced gene ex- Brassinosteroids have been identified in 27 families
pression, whereas the roles of BR in stress and of higher plants and three families of lower plants
(Bajguz and Tretyn 2003), and they occur in all
parts of higher plants, including roots. There are
Received: 30 April 2003; accepted: 15 August 2003; Online publication: 18
December 2003 some species differences, as comparison of the BRs
*Corresponding author; e-mail: [email protected] in Arabidopsis, pea, and tomato suggested three rate-

276
 Physiological Actions of BRs 277

limiting steps in their biosynthesis, and that the late terone was reported recently (Soeno and others
6-oxidation pathway a predominated in these di- 2000). Sulfation was confirmed, and expression of
cots. It was the only pathway in tomato and both the responsible sulfotransferase gene in Brassica na-
castasterone and the unusual BR, norcastasterone, pus was also induced by salicylic acid, suggesting
were present in comparable amounts (Nomura and that a modulation of steroid-dependent growth
others 2001). More detailed work on the reversible could be part of a plant’s response to pathogens
conversion of 24-epiteasterone to 3-dehydro-24- (Rouleau and others 1999). Such a dual role has
epiteasterone supported the involvement of at least also been proposed for a putative receptor in to-
two enzymes, with inhibition in the dark, suggest- mato, tBRI1/SR160, that recognizes both BR and
ing more mechanisms for ‘‘fine-tuning’’ of effective systemin (Montoya and others 2002). The two sig-
BR levels (Stündl and Schneider 2001). nal molecules do not compete directly, and Scheer
The particular cytochrome P450 enzymes in the and Ryan (2002) suggest that SR160 evolved from
biosynthesis of BRs are subject to temporal and BRI1, and that systemin signaling patterns were in
spatial in regulation, with feedback control by turn recruited as a means of storing proteins in
brassinolide concentration. In shoots and roots of developing potato tubers (Narvaez–Vasquez and
young seedlings there is organ-specific distribution Ryan 2002).
of transcripts, and concentrations of endogenous Another possibility for in vivo transport of BRs is
BRs correlate well with such expression in Arabid- the binding of BRs to protein carrier(s), which is
opsis, pea, and tomato (Bancos and others 2002). also relevant for signaling. Early work using im-
Another study in Arabidopsis, using two genes for C- munocytochemical techniques found no specific
6 oxidation in BR biosynthesis, BR6ox1 and BR6ox2, binding of brassinolide to any soluble protein ex-
and DWF4, showed expression primarily in young, tracted from pollen of rye grass (Smith and others
actively developing organs. Endogenous levels of 1992), but an NMR study suggested that 28-
BRs also correlated, with the greatest expression homocastasterone could bind to a cavity within an
and highest levels of endogenous BRs occurring in allergen from cherry (Neudecker and others 2001).
apical shoots, followed by siliques (Shimada and The major allergen from birch pollen, Bet v 1, is part
others 2003). A comprehensive review of the bio- of the PR-10 family of pathogenesis-related plant
synthesis and metabolism of BRs appeared this year proteins, and an isoform, Bet v 11, can form a
(Fujioka and Yokota 2003). complex with two deoxycholate molecules. Mass
However, we may ask: Is long-distance transport spectral analyses showed that brassinolide and 24-
of endogenous BRs important? It is well recognized epicastasterone also formed complexes (Markoviç–
that signaling molecules in plants and animals may Housley and others 2003). The authors proposed a
act over relatively long or short distances, and long- general plant-steroid carrier function for such pro-
distance transport of exogenous BRs can occur in teins that could be important in plant growth and
plants, particularly from root to shoot. Although development as well as defense responses. Such a
exogenous 24-epibrassinolide is not efficiently ex- role could be important even if biosynthesis of BRs
ported from leaves (Nishikawa and others 1994), it close to their sites of action proves to be more im-
is still possible that precursors might be important portant than long-distance transport.
for the transport of BRs in the whole plant. Local-
ized expression in particular cells of the leaves of a
gene encoding an enzyme important for the syn- EFFECTS IN DEVELOPMENT: CELL DIVISION
thesis of a crucial precursor to brassinolide (Mathur
and others 1998) could imply the presence of the Restoration of leaf size after administration of
complete biosynthetic pathway in those cells [with brassinolide to the mutants det2 and dwf1 of Ara-
export of the newly synthesized active BR(s) to the bidopsis could not be accounted for solely by ex-
sites of action] or export of precursor(s). Endog- pansion, supporting a role for BRs in cell division
enous BRs have been quantified in cell cultures of (Nakaya and others 2002). 24-epibrassinolide can
Zinnia elegans (Yamamoto and others 2001), and the substitute for cytokinin in the culture of Arabidopsis
relative quantities of particular BRs inside and callus and suspension cells, and a particular cyclin
outside the cells differed (discussed in the Cytodif- gene, CycD3, was upregulated. This also occurred in
ferentiation section). the BR-insensitive mutant bri1, reviving the possi-
Hydrolyzable conjugates may also be candidates bility of an unknown, possibly intracellular, path-
for transport. Glycosidation and esterification were way for the BR signal (Hu and others 2000).
reported some time ago (references in Bajguz and Constitutive overexpression of CYCD3; 1 affected the
Tretyn 2003) and reversible esterification of teas- anatomy of the leaf markedly, and a critical role has
278 J. M. Sasse

been proposed for this gene in the transition from Membrane permeability and selectivity in the
cell proliferation to differentiation (Dewitte and presence of toxic metals is also affected by BR, and
others 2003). the metal ions can be ranked in the order in which
The mitotic rate increased in roots of wheat after BR treatment ameliorated their effects. Results have
treatment with 24-epibrassinolide, and the hexap- been reported for selected crop plants (Khripach
loid Triticum aestivum was the most sensitive. Vol- and others 1999, 2000) and algal cells (Bajguz
umes of nucleoli were also increased, and the effects 2000b).
of cytokinin were similar (Fatkhutdinova and others Crucial for anisotropic growth in expansion are
2002). In synchronously dividing cultures of the polarization, axialization, and on-going control of
alga Chlorella vulgaris, accelerated increases in cell the orientation of microtubules and cellulose
number and marked increases in nucleic acid and microfibrils. Expression of b-tubulin genes corre-
protein levels followed BR treatment (Bajguz lated with brassinolide-induced growth in chickpea
2000a). Significant interactions with auxin at very (Munoz and others 1998), and, in Arabidopsis, BR
low concentrations of brassinolide were also ob- treatment restored elongation of the bul1-1 mutant.
served (A. Bajguz, personal communication), so this This severe dwarf, defective in the sterol synthesis
single-cell system has potential for exploring hor- pathway that leads to campesterol and BR, has
mone cross-talk and hormone-induced protein short, dissociated, and disordered microtubules
breakdown as well as species differences, if BRs (Catterou and others 2001a). Although TUB1 ex-
prove to be ubiquitous in algae. pression was affected, after consideration of their
data and current knowledge of the feedback rela-
tionships among microtubules, the plasma mem-
EFFECTS IN DEVELOPMENT: CELL brane, and the microfibrils of the cell wall, the
EXPANSION authors proposed that another ‘‘brassinosteroid-re-
sponsive pathway exists which allows microtubule
Whatever the molecular signals, expansion is con- nucleation/organization and cell elongation with-
tingent upon transport of ions, uncharged osmotica, out activation of tubulin gene expression.’’ They
and water across the cell and vacuolar membranes, also thought some anatomical characteristics of the
and upon a plastic cell wall. The possibility that BRs leaves of the bul1-1 mutant might reflect the lack of
may control aquaporin activities was explored using BR-induced TCH4 and expansin gene expression
the dwarf biosynthetic and sensitivity mutants cpd (Catterou and others 2001b). However, the possible
and bri1 of Arabidopsis. The median water permea- role of actin assembly and any role for BRs there
bility of wild-type protoplasts was significantly were not discussed.
greater than that of the mutants and covered a Also at the plasma membrane, because expres-
wider range of values. Brassinolide treatment in- sion was reduced in the Arabidopsis mutant det2, a
creased osmotic permeability of hypocotyl proto- role for BRs was suggested in the expression of KOR,
plasts from the cpd mutant markedly, with a shift in which encodes a membrane-bound endo-1,4-b-D-
the range of the population. As this was not a rapid glucanase unaffected by auxin, gibberellin, or eth-
response and the bri1 mutant was not affected, ylene (Nicol and others 1998). In rice, internodal
Morillon and coworkers (2001) concluded that elongation accompanies panicle formation, and, in
brassinolide was not likely to affect the membranes the internode, two xyloglucan endotransglucosy-
directly. However, mRNA and protein levels of lase/hydrolase genes, OsXTR1 and OsXTR3, were
aquaporin isoforms in root and shoot of radish were preferentially expressed in the elongating zone.
not affected by brassinolide treatment (Suga and These genes were upregulated by BRs and gibber-
others 2002). ellin, and Uozo and others (2000) suggested that
Seedlings of another Arabidopsis mutant, det3, BRs are essential for gibberellin sensitivity and that
develop as light-grown plants in the dark and have there is cross-walk between BR and gibberellin
reduced ability to respond to BR. The gene was signaling. Recent reviews of the roles of membrane-
shown to encode subunit C of the vacuolar H+– bound endo-1,4-b-D-glucanases (Mølhøj and others
ATPase, which has a role in the control of elonga- 2002), xyloglucan endotransglucosylation and
tion and also in meristem activity. The mutant has endohydrolysis (Rose and others 2002), expansins
an alternative method of assembly of this ATPase, (Cosgrove and others 2002), and cellulose biosyn-
and regulation of its activity by different signals thesis (Doblin and others 2002) in the construction
could occur through different configurations of the and modification of the cell wall illustrate that fur-
protein complex, with a BR signal transduced via ther elucidation of the effects of BRs in the complex
the DET3 protein (Schumacher and others 1999). relationships and information flow between micro-
 Physiological Actions of BRs 279

tubules, the plasma membrane, and the cell wall in EFFECTS IN DEVELOPMENT:
the process of expansion will be very valuable. CYTODIFFERENTIATION
Studies in other plants are also providing more
insights into the roles of BRs. Modification of the There is steady progress in the study of vascular
conditions in mesophyll suspension cultures of differentiation. Use of a potent inhibitor of BR bio-
Zinnia permitted the study of expansion only and synthesis, brassinazole, showed clearly that the de-
confirmed at the cellular level that 24-epibrassino- velopment of secondary xylem in Lepidum sativum
lide promoted elongation without significant radial was particularly affected, with normal development
expansion, in contrast to the effect of gibberellin. restored after administration of brassinolide (Nagata
Unlike the effects of light, the effects of hormones and others 2001). In Arabidopsis, a provascular/
began only after 48 h (Lee and others 2000). More procambial cell-specific gene, VH1, which marks the
extensive studies have been carried out in rice using transition to the procambial state, has been identi-
mutants in BR biosynthesis and response. Identifi- fied. Although it encodes a leucine-rich repeat re-
cation of the OsBRI1 gene provided evidence for its ceptor kinase, it does not respond to 24-
involvement in internode elongation, asymmetric epibrassinolide (Clay and Nelson 2002). This is
growth in the lamina joint, and skotomorphogene- consistent with data from the model system of cul-
sis. Internode elongation depended on the BR signal tured Zinnia cells suggesting that BRs are most im-
for the formation of the intercalary meristem, and portant in the later stages of vascular development
internodes differed in their sensitivity. More OsBRI1 (Yamamoto and others 1997). In Zinnia, endog-
was expressed in dark-grown rather than in light- enous BRs were shown to increase markedly before
grown seedlings, which is appreciably different than stage III of the differentiation process, where sec-
BRI1 in Arabidopsis (Yamamuro and others 2000). ondary wall formation and cell death occur. Seven
The asymmetric expansion occurring in rice lamina BRs were monitored; levels of castasterone, typha-
inclination in response to brassinolide involves a sterol, their 6-deoxo-analogues, and 6-deoxo-teas-
calcium-dependent protein kinase (Yang and terone increased, with the changes in 6-deoxo-
Komatsu 2001), which may also hint at species castasterone and 6-deoxo-typhasterol the most
differences, whereas Sharma and others (2001), in a marked. The 6-deoxo-sterols were far more abun-
study of mitogen-activated protein kinase and cal- dant than the 6-oxo-BRs, but the latter were pref-
cium-dependent protein kinase in BR signaling in erentially excreted into the medium. The authors
rice, suggested, like Hu and others (2000), that propose a signaling pathway whereby extracellular
there are other BR receptor(s) besides BRI1 in BRs are detected by a surface receptor like BRI1,
plants. and a signal cascade follows. The final stage of dif-
Two groups have reported rice mutants with de- ferentiation is then initiated (Yamamoto and others
fects in BR biosynthesis, both at a late 6-oxidation 2001) and the processes of lysis of the vacuole and
step. A severe dwarf, brd1, had marked phenotypic modification of the tracheary elements follow
changes when grown in light and constitutive (Kuriyama and Fukuda 2002). Any effects of BR on
photomorphogenesis in the dark. Data for roots the process of lignification are, as yet, unknown.
suggested BR promotes the development of crown In young Zinnia plants, mRNA transcripts of ho-
roots, primary and secondary branched roots, and meobox genes (analogous to those coding for Class
the extension of thin primary branched roots. Study III HD-Zip proteins in Arabidopsis) accumulated in
of the leaf sheaths and blades suggested effects on procambium and immature xylem. In cultured cells,
both expansion and division, but exogenous bras- expression was suppressed by a selected inhibitor of
sinolide could not restore reproductive develop- BR biosynthesis but was restored by brassinolide.
ment (Mori and others 2002). The other group The authors concluded that the encoded proteins
(Hong and others 2002) described the anatomy in could be transcriptional regulators of xylogenesis,
three allelic mutants with very similar phenotypes acting downstream from the BR signal. Because
to that of Mori and others (2002). Introduction of members of this class of genes have a sterol binding
the wild-type gene restored the normal phenotype, domain, they also stressed the importance of iden-
expression analysis showed the greatest intensity in tifying such a ligand and did not exclude a BR as a
the leaf sheath, and C-6 oxidase activity was con- candidate (Ohashi-Ito and others 2002). However,
firmed. The authors consider that their data reflect sterols other than the BR series are potential signal
an underlying disarray of the microtubules in the molecules (summarized by Clouse 2002b). The CVP1
mutants, and also think that the lack of BRs may gene encodes a sterol methyltransferase (SMT2),
affect determination of sites for cell division (Hong which acts where BR biosynthesis diverges from the
and others 2002). main route to sterols such as sitosterol and stigma-
280 J. M. Sasse

sterol. In Arabidopsis, the mutant cvp1 has disor- EFFECTS IN DEVELOPMENT:

ganized vascular axialization and misshapen vas- VEGETATIVE GROWTH
cular cells in the cotyledons, and it accumulates
early precursors to BRs, but the phenotype does not The use of inhibitors of brassinosteroid biosynthesis
reflect high endogenous levels of the most active and exogenous application of BRs to whole plants
BRs. So Carland and others (2002) proposed that and explants continue to provide useful leads for
other signaling sterols control vascular cell polari- more detailed studies. For instance, it is well known
zation and axialization. Thus, there are promising that BR can enhance ethylene biosynthesis, and
leads for better understanding of the whole process root treatment of Arabidopsis seedlings with brassi-
of vascularization and the undoubtedly complex nolide or its 24-epimer did do this, but a specific
interactions between all the chemical signals in- upward rather than a downward curvature of that
volved. part of the petiole nearest the leaf was observed. An
ethylene-insensitive mutant responded similarly,
suggesting that the effect was independent of eth-
EFFECTS IN DEVELOPMENT: ylene, as well as of the particular auxins and gibb-
SEED GERMINATION erellin that were also tested (Arteca and Arteca
2001). Could this result point towards an endog-
Using exogenous applications of brassinolide and enous effect of BRs in phototropism? Is there a role
gibberellin, Leubner–Metzger (2001) found differ- for BRs in the hyponastic response of Rumex palustris
ing effects on tobacco seed germination depending to flooding (Peelers and others 2002)?
on the state of dormancy, or on whether imbibition Experiments with whole plants also interest those
occurred in the dark or light, or on whether the hoping to maximize or accelerate crop yield. For
seeds were inhibited by abscisic acid. The author example, a spray treatment of mustard seedlings
concluded, from effects on endosperm rupture and with 28-homobrassinolide led to higher fresh and
the induction of a b-1,3-glucanase in the micropylar dry weights, enhanced carbonic anhydrase activity
endosperm, that the two hormones acted in distinct and net photosynthetic rate, and enhanced pod
pathways. He proposed that gibberellin and light act number and seed yield per plant at harvest (Hayat
in a common pathway, whereas BR directly en- and others 2000). Comparison of the effects of the
hances the growth of the emerging embryo inde- optimal concentration (10 nM) with those of lM
pendent of gibberellin and that particular b-1,3- levels of indole-3-yl-acetic acid, gibberellic acid,
glucanase. In Arabidopsis, Steber and McCourt kinetin, and abscisic acid showed that the BR was
(2001) found 24-epibrassinolide and brassinolide the most promotive of the regulators and that
treatment rescued mutants whose gibberellin bio- chlorophyll levels were also enhanced (Hayat and
synthesis is severely inhibited, and one which was others 2001a). However, in soybean seedlings, only
gibberellin insensitive. They also found that germi- inhibitory effects on root and shoot length, dry
nation of both a BR-biosynthetic and a BR-insen- weight, and nodule and lateral root numbers were
sitive mutant (det2-1 and bri1-1) was inhibited more observed after roots were treated with 0.1–10 lM
strongly in the presence of abscisic acid than the 24-epibrassinolide (Hunter 2001). Although 24-
wild type. They suggest that endogenous BR as well epibrassinolide is regarded as less active than bras-
as gibberellin are needed to overcome ABA-induced sinolide, the concentrations for root treatments do
dormancy and that BR acts in parallel or down- seem relatively high; effects of lower concentrations
stream of gibberellin. could be of interest. Using brassinolide itself, Chon
A more developed model comes from the work of and others (2000) found that 11 representative
Ullah and others (2002), who considered whether cultivars of rice had increased leaf sheath lengths
coupling and cross-talk among ethylene, glucose, and numbers when grown in light, whereas pre-
abscisic acid, gibberellin, and BR signals in germi- treatment of seeds enhanced mesocotyl elongation
nation involve heterotrimeric G proteins. Using in the dark. Some inhibitory effects were observed
Arabidopsis mutants det2-1 and bri1-5, they at- at the highest concentration used.
tempted to distinguish direct coupling via the G- Wheat grown from seed treated with lM con-
protein complex or potentiation by alteration of centrations of 28-homobrassinolide had enhanced
sensitivity to a particular signal. Regarding brassi- leaf number per plant, fresh and dry weight, and
nolide and its effect on the mutant gpa1, they con- nitrate reductase and carbonic anhydrase activity
cluded that BR might be coupled via GPA1 to (Hayat and others 2001b). In another study in
potentiate the gibberellin-controlled pathway for wheat, pretreatment of seeds with 24-epibrassino-
germination. lide at concentrations ranging from 0.04 nM to 40
 Physiological Actions of BRs 281

lM led to a biphasic promotion of root length, with engineering of other species is considerable, and
inhibition evident only after treatment with the effects in woody species will be fascinating.
highest concentration of the BR. The content of
agglutinin was elevated in the most elongated roots,
without a concomitant increase in abscisic acid EFFECTS IN DEVELOPMENT:
concentration, but any role for this protein in
elongation is not yet understood (Shakirova and
APICAL DOMINANCE, REPRODUCTION,
others 2002). With rice, time of application and AND SENESCENCE
length of exposure to brassinolide were important.
Shoot lengths of resulting seedlings were signifi- Although disturbances in apical dominance, bolting,
cantly promoted seven days after continuous and flowering, silique yield, and timing of senescence in
first day treatments of the seeds but not after Arabidopsis have been observed in molecular genetic
treatment on days 3 and 4 of germination. Root studies utilizing mutants, more focused studies on
elongation varied dramatically from enhancement BRs’ effects in these areas have rarely been reported
to inhibition (Fujii and Saka 2001a). in the last few years. Recent work includes a study
Whole plants, with their need for coordination of embryogenesis, where BRs, although crucial for
between organs and their coordinated response to postembryonic growth, were not so for cell prolif-
their environmental conditions, challenge our eration, but other sterols were important (Shrick
concepts of control and complexity. In an opinion and others 2002).
paper, Amzallag (2001) questioned the assumption A study of ripening of pericarp discs from tomato
that determination of variability in physiological showed lycopene and carbohydrate levels and eth-
studies was useful only for testing differences be- ylene production increased, whereas levels of
tween means for significance. Amzallag proposed chlorophyll and ascorbic acid decreased, after
analyzing variability and using it for the investiga- treatment with 24-epibrassinolide or 28-homo-
tion of redundancy and patterns of control in bio- brassinolide, which is consistent with accelerated
logical systems; connectance, coefficients of ripening (Vardhini and Rao 2002). In rice, brassi-
variation (CV), and differences between calculated nolide treatment before and at heading also accel-
ratios were discussed. As an example, in roots of erated ripening and significantly increased starch
Sorghum bicolor seedlings treated with 0.1–10 nM content in hulled grain (Fujii and Saka 2001b).
24-epibrassinolide, highly significant increases in During a search for senescence-associated genes,
shoot and root fresh weight, and in the length of He and others (2001) developed a preliminary
the sheath and blade of the fifth leaf, were observed model for a leaf senescence-regulating network in
only for the plants treated with 10 nm 24-epibras- Arabidopsis where signals such as abscisic acid, jas-
sinolide. There was no clear effect on variability, monic acid, ethylene, darkness, dehydration, and
but in total plant data for those treated with the aging activated 147 senescence-associated enhancer
lowest concentration of BR, the CV value appeared trap lines. 24-epibrassinolide could activate two of
reduced. A comparable experiment (Table 1, J. these but associated genes have not yet been cloned.
Sasse unpublished) showed no such trend, and
there was a wide range of values for the CVs within
the initial values where the means were not sig- ENDOGENOUS INTERACTIONS
nificantly different. One suspects Amzallag’s aims
would be better served by testing with increased Some progress has been made in the study of
replication and expert statistical input. However, source–sink relations. Early work with exogenous
the data for the highest concentration of brassino- BRs in bean had suggested that BRs (and auxin and
lide in Table 1 are consistent with Gregory’s work gibberellin) enhance sink strength and phloem un-
(1981) where brassin treatment reduced pheno- loading (Petzold and others 1992), and Nakajima
typic variability. and Toyama (1999) showed treatment of cucumber
A more conventional approach exploited knowl- roots with 24-epibrassinolide promoted transport of
14
edge of the biosynthetic mutant dwf4 of Arabidopsis. C-labeled sucrose from the primary leaf to the
Overexpression of DWF4 produced a dramatic in- epicotyl. Fujii and Saka (2001b) monitored the ef-
crease in hypocotyl length in both light and dark fect of brassinolide treatment on the distribution of
and in inflorescence length at maturity. The num- starch and sucrose to organs of rice plants. Starch
ber of branches and siliques also increased, with a accumulated in the grains at the expense of the leaf
concomitant increase in seed yield (Choe and others sheaths and culms, whereas sucrose levels de-
2001). As the authors suggest, the potential for creased in the culms.
282 J. M. Sasse

Table 1. Mean Heights and Coefficients of Variation of Radish Seedlings after Root Treatment with
Brassinolidea

BR Treatment nM 0 0.1 1 10 100

S Initial mean 7.7 7.4 7.6 7.1 7.5
Overall mean 7.46 mm
CV 12.3 20.3 19.9 8 11.3
Overall CV 14.9
L Initial mean 20.5 18.3 18.8 18.6 17.8
Overall mean 18.8 mm
CV 13.8 15.9 13 9.9 7.4
Overall CV 12.9
S Final mean 30.8 34.7 32.9 32.4 45.6*
CV 20.1 36.4 22.4 25.3 25
L Final mean 45.1 42.4 43.3 45 52*
CV 18.5 26.4 18.7 17.4 21.5

a
One hundred fifty 4-day-old seedlings from a single batch were divided equally into small (S), medium, and large (L) groups. Plants
within the groups were randomly allocated to 5 hormone treatments. Initial mean heights within the S and L groups were not
significantly different (n = 10). Seedlings were grown hydroponically for 4 days.
*These values were not significantly different (p = 0.05).

Extracellular invertases are important for the response curves that reflect additive or synergistic
supply of carbohydrate to sink tissues, and in to- responses with other hormones were shown for
mato cell culture, treatment with three BRs led to a brassinolide (Sasse 1989, 1990). Such studies can
specific enhancement of cell-wall-bound invertase point to experimental systems where more detailed
activity, with a rapid induction of the mRNA for one investigations using current and developing tech-
isoenzyme, Lin6. As expected, sucrose uptake was niques could provide valuable information.
also enhanced, dependent upon the enhanced in- A molecular genetic approach (together with ro-
vertase activity. Furthermore, in hypocotyl seg- bust analysis of data) was illustrated in an interac-
ments, BR-dependent growth ‘‘correlated with a tion between BR and abscisic acid in Arabidopsis,
localized, tissue-specific induction of mRNA for where a model has been developed for positive
extracellular invertase Lin6’’ (Goetz and others regulation of three genes, BEE1, BEE2, and BEE3, by
2000). Thus, BRs not only promote elongation but BR, with negative regulation by abscisic acid. These
also, like other hormones, help coordinate the genes encode transcription factors important in
supply of carbohydrate necessary for that and other ‘‘several developmental programs, including floral
responses (Roitsch and others 2003). organogenesis, hormone responses, and light sign-
It is well recognized that BRs influence various aling through phytochrome’’ (Friedrichsen and
developmental pathways. Such effects have their others 2002). BR treatment can also inhibit ex-
own feedback controls and inevitably impinge on pression of, for example, the abscisic acid-induced
other processes and interact with other influences. gene P5CS1, important for the synthesis of the os-
These might be other hormones and macromolec- moprotectant proline (Abrahám and others 2003).
ular signals, internal conditions at the cellular and Another example of an interaction between BR and
organ level such as pH, and ion and metabolite abscisic acid was discussed in the Seed Germination
concentrations, as well as external conditions. section.
Studies of such interactions have examined changes Regarding hormone levels, pretreatment of Ara-
in endogenous levels of other hormones, described bidopsis seeds with 1 lM 24-epibrassinolide led to
synergistic effects with exogenous treatments, and considerable cotyledon expansion and slight inhi-
discussed modulations of sensitivities of response. bition of hypocotyl elongation of dark-grown seed-
For example, increased sensitivity to BRs in dark- lings which were accompanied by significant
grown rice (Yamamuro and others 2000) could re- elevations of free and bound auxin and of free ab-
flect increased numbers of BR receptors. Quite so- scisic acid, a slight elevation of free cytokinin, and a
phisticated kinetic approaches have been used decrease in bound abscisic acid (Karnachuk and
occasionally (Weyers and others 1995, and refer- others 2002). Such changes could alter cellular ex-
ences cited therein); significant differences between pansion, and possibly ethylene signaling, and may
 Physiological Actions of BRs 283

imply that BRs upregulate the biosynthesis of other the perception of BR by BRI1. However, a full re-
hormones; for example, an effect of BR on the ex- sponse to auxin does require it (Iliev and others
pression of an important enzyme for the biosyn- 2002).
thesis of jasmonic acid has been reported (Schaller Evidence that BR treatment can ameliorate vari-
and others 2000). However, BRs might also impinge ous biotic and abiotic stresses in plants was dis-
on their catabolism, and, in the case of auxin where cussed by Khripach and others (1999, 2000) and by
marked synergistic effects can occur in the presence Krishna in this issue. They and others emphasize
of both hormones, BRs might affect auxin-depend- the importance of the induction of antioxidant en-
ent targeted protein degradation (reviewed in Ley- zymes to protect cells, for example, in temperature
ser 2001). On the other hand, as Ullah and others stress (Mazorra and others 2002). Brassinolide
(2003) proposed, BRs might modulate auxin action treatment enhanced early seedling growth, grain
upstream of its transcriptional control by coupling ripening, and lamina inclination in chilling stress in
via AGB1 in a heterotrimeric G-protein complex. rice (Fujii and Saka 2001ab), and Yu and others
(2002) found that pretreatment with 24-epibrassi-
nolide or abscisic acid increased tolerance and
photosystem II efficiency in cucumber. Pretreat-
INTERACTIONS WITH ENVIRONMENTAL ment of seeds with a formulation of synthetic BR
SIGNALS AND STRESSES and jasmonic acid derivatives also protected cu-
cumber (Asao and others 2002).
Tropisms, particularly geotropism in stems, received In salt stress, pretreatment of rice seeds with 24-
some attention in the early years of BR research, epi- or 28-homobrassinolide promoted germination
and, in a more recent study, exogenously supplied in the presence of sodium chloride. Lengths, fresh
brassinolide rapidly enhanced geotropic curvature and dry weights, and DNA, RNA, and soluble
of maize roots. There was a clear interaction with protein contents of the resulting seedlings were also
auxin, whose transport was essential for the re- enhanced (Anuradha and Rao 2001). The soluble
sponse. Castasterone, although not as effective as protein levels were even greater than those of the
brassinolide exogenously, was identified endogen- water control; one hopes these can be investigated
ously, and Kim and others (2000) suggest that BR in more detail. Also, treatment with a BR analogue
increases the sensitivity of the responding tissue to enhanced antioxidant enzyme activity in rice ex-
auxin. posed to salt (M. A. T. Zullo, personal communica-
There has also been longstanding interest in the tion). However, in Arabidopsis, expression of a salt-
interaction of BRs with light signals, and recent induced reporter gene, P5CS1-GUS, was inhibited by
advances have been reviewed (Clouse 2001). Fine brassinolide treatment (Abrahám and others 2003).
tuning (Luccioni and others 2002) and species dif- There is renewed interest in the protective effects
ferences have been emphasized (Kang and others of BR on plants under pathogen attack. Results from
2001; Symons and others 2002; Symons and Reid rice and tobacco plants infected with representative
2003). The effects of green light were synergistic viral, bacterial, and fungal pathogens showed that
with those of BR in wild-type Arabidopsis (Karna- biosynthesis of salicylic acid was not required, and
chuk and others 2002), and bioassay data suggested acidic and basic pathogenesis-related protein syn-
that endogenous BR levels are markedly increased thesis was not induced in BR-induced resistance
when rice seedlings are exposed to blue light, a (which may also provide another example of species
signal that is particularly promotive of the unrolling differences from Arabidopsis). The data suggest that
of etiolated leaves (Abe and others 2000). However, BR-induced resistance, though ‘‘moderately weak,’’
there is still very little information about any effects can be distinguished from systemic acquired resist-
of BRs on phototropism, photoperiodism, or en- ance and wound-inducible resistance, and that it
dogenous rhythms. might provide added protection (Nakashita and
Similar responses to diverse stimuli and redun- others 2003).
dancy are concepts that are difficult to explore ex-
perimentally, but some progress is being made. The
Arabidopsis TCH4 gene, which encodes an endo- OTHER PHYSIOLOGICAL EFFECTS
transglucosylase hydrolase, is upregulated by envi-
ronmental stimuli as well as hormones, and recent Effects of BRs on insect development, particularly
work suggested that responses to darkness, cold, molting, were reviewed by Zullo and Adam (2002).
heat, and BRs occur via shared regulatory elements, Exploration of the effects of BRs in model systems
and that the environmental stimuli do not require has provided insights into the catabolism of these
284 J. M. Sasse

compounds, but prospects for their use in predation 23S)-homobrassinolide (Wang and others 2002). BR
control do not seem imminent. For example, 24- analogues were also used to accelerate vegetative
epibrassinolide or 24-epicastasterone did not affect bud formation in cactus pear, increasing the num-
the evagination of imaginal wing discs, nor was ber of cladodes harvested as well as total fresh
there any effect on intact last-instar larvae of the weight (Cortes and others 2003).
cotton leafworm, Spodoptera littoralis, after oral Interest in delaying sprouting of potatoes during
feeding (Smagghe and others 2002). However, storage also continues. The dosage of 24-epibrassi-
structurally modified BRs may prove more useful. nolide is important, and enhanced evolution of
The low toxicity and low mutagenicity of BRs has ethylene and increased abscisic acid content (with a
been confirmed and protective effects in fish change in the ratio of free to bound abscisic acid)
breeding reported. Beneficial effects of BRs for hu- followed exogenous treatment with the most ef-
mans were also postulated; apart from our routine fective concentration. The main target cells were
ingestion of ubiquitously occurring BRs in plants, those of the rib meristem; inhibition of their elon-
the claimed health benefits of pollen may be related gation is of primary importance for delaying
to the BR content (Khripach and others 1999, sprouting (Korableva and others 2002). Appropriate
2000). There are some studies of antiviral effects; formulation is required for the most effective use of
analogues of 24(S)-ethylbrassinone were tested exogenously applied BRs; wetting agents and addi-
against herpes simplex virus type 1 and arena vi- tives that slow evaporation and assist penetration
ruses (Wachsman and others 2000) and structure are useful, and these aspects have been discussed for
and activity were correlated against the measles crops by Khripach and others (1999, 2000). The use
virus (Wachsman and others 2002). of inclusion complexes is also being explored. At the
lowest concentrations used, a 1:1 inclusion complex
between 24-epibrassinolide and b-cyclodextrin was
TOWARDS APPLICATIONS more effective on sensitive cultivars in the rice
lamina inclination bioassay than free 24-epibrassi-
The need for efficient crop production, land regen- nolide (de Azevedo and others 2002; M. A. T Zullo,
eration, and reafforestation in the context of global personal communication). In vivo, endogenous BR is
problems of salinity, drought, rising temperature, associated with starch granules in pollen and is
pollution, and pathogen and predator attack is ob- easily leached during aqueous fixation (Taylor and
vious, so exploration of BRs’ potential for applica- others 1993).
tions continues. Recently, Zullo and Adam (2002) Triadimefon is a widely used fungicide with
summarized prospective agricultural uses of BRs useful side effects of dwarfing, delay of senescence,
and some of their analogues. and increased greening in plants. Arabidopsis plants
One concern is improvement of propagation treated with Triadimefon were rescued by combi-
methods, not only for conventional commercial nations of gibberellin and BRs and the fungicide
applications but also if we are to utilize our rapidly bound with expressed DWF4 protein. The biosyn-
increasing number of useful transformed plants. thetic pathway to active BRs was inhibited, induc-
Treatment with an analogue of 28-homoethylca- ing a BR deficiency. Specific inhibitors of the
stasterone enhanced the production of shoots on biosynthesis of BRs may be useful additions to the
nodal segments from shoots of Malus prunifolia, al- repertoire of pesticides and safeners used in agri-
though the dosage was critical. Both stem elonga- culture and horticulture (Asami and others 2003).
tion and lateral branching were increased, with the These data may also renew interest in the effects of
secondary branching more variable than the pri- BRs on fungal development (reviewed by Zullo and
mary (Schaefer and others 2002). Other analogues Adam 2002). Another idea for future work could be
of BR enhance callus formation and shoot regen- the protective effect of BRs on division of Chlorella
eration in lettuce in conjunction with cytokinin (M. vulgaris in the presence of toxic metal ions, and their
A. T. Zullo, personal communication). Adventitious acceleration of the early stages of endogenous
shoot regeneration in response to brassinolide itself phytochelatin synthesis induced by lead nitrate
has been reported in cauliflower hypocotyl seg- (Bajguz 2000b, 2002)
ments, and the response was markedly enhanced in
the presence of appropriate doses of cytokinins
(Sasaki 2002). Levels of the secondary metabolite, OTHER SOURCES
artemisinin, as well as biomass, DNA, RNA, and
soluble protein were increased by treatment of a Further studies, for example, in intracellular im-
hairy root culture of Artemisia annua with (22S, munolocalization of brassinolide, were reported in
 Physiological Actions of BRs 285

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