Brassinosteroids. Chemistry, Bioactivity, and Applications

357 Pages·1991·7.89 MB·English

by  Horace G. Cutler

ACS SYMPOSIUM SERIES 474
Brassinosteroids
Chemistry, Bioactivity, and Applications
Horace G. Cutler, EDITOR
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00 U.S. Department of Agriculture
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1- Takao Yokota, EDITOR
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k-1 Teikyo University (Utsunomiya, Japan)
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10. Günter Adam, EDITOR
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d Institute of Plant Biochemistry (Halle/Saale, Germany)
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o Developed from a symposium sponsored
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e: by the Division of Agrochemicals
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D at the 200th National Meeting
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atio of the American Chemical Society,
blic Washington, D.C.,
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P August 26-31, 1990,

and an international workshop sponsored
by the Federation of European Chemical Societies
at the Institute of Plant Biochemistry,
Halle/Saale, Germany,
October 29-November 2, 1990
American Chemical Society, Washington, DC 1991
Library of Congress Cataloging-in-Publication Data
Brassinosteroids: chemistry, bioactivity, and applications / Horace
G. Cutler, editor, Takao Yokota, editor, Gunter Adam, editor.
p. cm.—(ACS symposium series; 474)
"Developed from a symposium sponsored by the Division of
Agrochemicals at the 200th National Meeting of the American Chemical
Society, Washington, D.C., August 26-31, 1990, and an international
workshop sponsored by the Federation of European Chemical Societies
at the Institute for Plant Biochemistry, Halle/Saale, Germany, October
1
0 29-November 2,1990."
0
w
4.f Includes bibliographical references and index.
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1-0 ISBN 0-8412-2126-X
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1 1. Brassinosteroids—Congresses.
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1/ I. Cutler, Horace G., 1932- . II. Yokota, Takao, 1942- . III.
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0 Adam, Gunter. IV. American Chemical Society. Division of
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0. Agrochemicals. V. Federation of European Chemical Societies.
oi: 1 VI. Series.
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91 | Q58K18.1998-2.B78—5Bdc7230 1991 91-31405
19 CIP
4,
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ov The paper used in this publication meets the minimum requirements of American National
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n Copyright © 1991
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PRINTED IN THE UNITED STATES Of AMERICA
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1991 ACS Books Advisory Board
V. Dean Adams Bonnie Lawlor
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Foreword
THE ACS SYMPOSIUM SERIES was founded in 1974 to provide
a medium for publishing symposia quickly in book form. The
format of the Series parallels that of the continuing ADVANCES
IN CHEMISTRY SERIES except that, in order to save time, the
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w0 ted by the authors in camera-ready form. Papers are reviewed
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Preface
BRASSINOSTEROIDS, a new group of biologically active natural prod­
ucts, are under consideration as another class of phytohormones. Their
discovery and development mark a major milepost in the field of plant
growth regulators, and have had a profound effect on plant physiology
and agronomy. These natural products are also of interest to other disci­
1 plines, such as mycology, entomology, horticulture, and (perhaps in the
0
0
pr future) even mammalian physiology. The potential economic effects
4.
7 these environmentally benign compounds may have on grain crop yields
4
0
1- are immense.
9
19 The original discovery of brassinolide, a natural plant-growth promoter
k-
b extracted from bee-collected pollen, was made at the USDA Agricultural
1/
2 Research Center in Beltsville, Maryland. Although the USDA terminated
0
1
0. brassinosteroid research, others around the world have carried on the
1
oi: work. This volume addresses the history, biochemistry, physiology, practi­
d
1 | cal applications, production, synthesis, and entomological effects of these
99 compounds.
1
4, As the product of significant international effort, this book offers a
ber compilation of the current work in the field. To date, it is the only
m
ve comprehensive review of the subject. Without the generous support of
o
N the Division of Agrochemicals of the American Chemical Society and the
e:
at substantial help of Maureen Rouhi, the book would have been impossible.
D
n We also thank BASF AG, Hoechst AG, Firmenich AG, Mandaus AG,
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ati Schering AG, Shell Agrar AG, and Stickstoffwerke AG Piesteritz which
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bli generously supported the Halle workshop. Without the effort of all these
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P people, this volume would not have been possible.
HORACE G. CUTLER GÜNTER ADAM
Richard B. Russell Research Center Institute of Plant Biochemistry
Agricultural Research Service Weinberg 3
U.S. Department of Agriculture Halle/Saale, O-4050, Germany
Athens, GA 30613
TAKAO YOKOTA
Department of Biosciences
Teikyo University
Utsunomiya 320, Japan
July 22, 1991
Chapter 1
U.S. Department of Agriculture Brassins
Project: 1970-1980
G. L. Steffens
Beltsville Agricultural Research Center, Agricultural Research Service, U.S.
Department of Agriculture, Beltsville, MD 20705
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0
h
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4.
47 The discovery of a new naturally occurring group
0
1- of plant growth promoting substances, termed
9
9 brassins, from various sources of pollen was
1
k- reported in 1970 (Nature 1970, 225:1065). In 1974
b
1/ a cooperative effort for identifying the active
2
0 component(s) of brassins was initiated among
1
0. U.S.D.A. scientists at the Northern Regional
1
oi: Research Center (NRRC), Peoria, IL; the Eastern
91 | d RPAe;g ioannadl Rthees eaBrcehl tsCveinlltee r A(EgRriRcCu)l, tuPrhali laRdeeslpeahricah,
9
1 Center (BARC), Beltsville, MD. To obtain enough
ber 4, c2r2u7d ek gp oolfl ebne ee-xctorlalcetc tefdo r riadpeen t(iBfircaastsiiocan, naabpouust L.)
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e pollen was processed via a pilot plant-size
v
o solvent extraction procedure at ERRC and
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e: partially purified at BARC (Ind. Eng. Chem. Prod.
Dat Res. Dev. 1978, 17:351). A few crystals (4 mg) were
n obtained at NRRC and subjected to x-ray
o
ati crystallographic analysis to determine structure
blic (Nature 1979, 281:216). This biologically active
u plant growth promoter, brassinolide
P
[2α, 3α, 22α, 23α-tetrahydroxy-24α-methyl-β-homo-7-
oxa-5α-cholestan-6-one], was found to be a
steroidal lactone with an empirical formula of
C H O (MW = 480). The concentration of
b2r8a4s8s6inolide in rape pollen is estimated to be
about 100 parts per billion. Syntheses of
brassinosteroids, biologically active analogues
of brassinolide, were first conducted at BARC (J.
Organic Chem. 1979, 44:5002) .
The structure of the unique biologically active plant
growth promoter, brassinolide, from pollen of the rape
plant (Brassica napus L.) was published in 1979 (I). That
This chapter not subject to U.S. copyright
Published 1991 American Chemical Society
1. STEFFENS USDA Brassins Project: 1970—1980 3
publication was the culmination of more than 10 years work,
at first by a few USDA scientists at Beltsville, MD and
later by a multi-disciplinary team at three USDA locations:
The Northern Regional Research Center (NRRC), Peoria, IL;
The Eastern Regional Research Center (ERRC), Philadelphia,
PA; and The Beltsville Agricultural Research Center (BARC) ,
Beltsville, MD. This Chapter presents an historical
perspective of the research effort that led to the
isolation and identification of brassinolide and to the
first synthesis of brassinosteroids, the group of compounds
that are structural isomers of brassinolide (2). General
aspects of the project, along with details of the chemistry
of natural and synthetic brassinosteroids have recently
been published (3) . Additional information on biological
and chemical properties of brassinolides will be presented
01 in the Chapters that follow.
0
h
c
4. Prior to 1970 - BARC, Beltsville, MD
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4
0
1- Early in his career, J.W. Mitchell (ret.) was interested in
9
19 the possibility of finding plant hormones and growth
bk- substances in extracts of pollen (4) and immature seeds
21/ (5). The rationale for this was the fact that such plant
10 tissues have the capacity for rapid growth which most
10. likely is stimulated or triggered by specific hormones or
oi: growth substances. In order to determine if plant growth
d
1 | inhibiting or promoting substances reside in these or other
9 plant tissues, active components needed to be extracted, at
9
4, 1 least partially separated from impurities, and then
er subjected to some type of biological assay system.
b Mitchell sought out and developed plant bioassay systems
m
e for the purpose of evaluating and documenting specific
v
No responses to known and newly discovered plant hormones and
e: growth substances (6) .
at
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on 1970 to 1974 - BARC
ati
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bli Mitchells search for biological activity in pollen led in
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P 1970 to the first published report on the biologically

active fraction, termed brassins, from an ethyl ether
extract of pollen of the rape plant (Brassica napus L.) (7).
The partially purified biologically active extract from
rape pollen will be referred to as "brassins" in this
Chapter. The extract was partially purified via a thin
layer silica gel chromatographic procedure. Biologically
active fractions were detected and monitored via the "bean
2nd internode bioassay" (6) . Fractions from the
chromatogram were mixed with a carrier (fractionated
lanolin) and applied, with the aid of a dissecting
microscope, to 1 mm long 2nd internodes of bean plants
(Phaseolus vulgaris L., cv. Pinto). Plants were grown for
four days under 700 foot-candles of fluorescent light (9
hr. photoperiod) in a room maintained at 20°-23°C and then
the internodes were measured. The initial report indicated
4 BRASSINOSTEROIDS: CHEMISTRY, BIOACTIVITY, AND APPLICATIONS
that ...."brassins induced very marked elongation of both
second and third internodes of the intact plants when
applied at the rate of 10 iiq per plant. For example,
second internodes grew an average of 155 mm during four
days immediately after treatment, while controls treated
with the fractionated lanolin alone grew only 12 mm." (7).
Histological studies showed that the response to brassins
was different from the response induced by gibberellic
acid. Reported chemical and physical properties of
brassins suggested that it was a "family" of new plant
hormones that appeared to have glyceride structures.
Further studies of pollen extracts of a number of
different plant types for brassins-like activity showed
that some induced the same type activity whereas others did
not, and some were inactive (8, 9) . Because rape pollen
1 was a rich source of brassin-like activity, and because it
0
h0 could be relatively easily obtained, it was chosen for
c
4. further detailed chemical and biological studies.
7
4 Along with the bioassay studies, an active program to
0
1- investigate the physiological properties of brassins was
9
19 being pursued. Worley and Mitchell (10) showed that
k- brassins accelerated growth of the treated internodes, as
b
1/ well as internodes above and below the treated area, in
2
10 beans. These responses involved both cell elongation and
10. cell division which caused marked changes in the vascular
oi: anatomy of leaves above the site of brassins application.
91 | d Srteusdpioensd ebdy Wtoo rlberya sasnidns Krtirzeeka tsmheonwt edm utchha t twhoeo dsya pmle anatss baelsaon
9
1 plants (11) and that light influenced the way bean
er 4, internodes responded to brassins (12). More controversial
b was the report by Mitchell and Gregory (13) that brassins
m
e increased the overall growth of plants. These findings
v
No were the basis for postulating that brassins may function
e: by providing hormonal control at fertilization for
Dat accelerating and enhancing overall plant growth and
n development - the "alpha hormone" function. Criticism (14)
o
ati was also directed at the apparent lack of detailed chemical
blic and physical information on the active component(s) of
u brassins.
P

Although there was no formal response to the published
criticism, much effort was devoted to answering some of the
questions posed and to evaluating the effects of brassins
on intact plants under both greenhouse and field
conditions. Unpublished data [Mitchell, J.W., USDA, (ret.)
personal communication, 1973] obtained from several
cooperative experiments in different parts of the country
suggested that field-grown cereal and vegetable crops
responded to brassins treatment by producing larger seed
yields and/or larger plants. Many detailed greenhouse and
growth chamber studies were also conducted at Beltsville
based on the concept that brassins stimulated plants to
express, more fully, their growth potential. This then
allowed brassins-treated plants to attain full development
sooner, provided the environment was suitable (15) .
1. STEFFENS USDA Brassins Project: 1970—1980 5
Because of the need for larger amounts of brassins,
one phase of the chemistry program during this period
centered on a convenient, practical, and reproducible
method for their production from dried rape pollen. The
preparative thin- and thick-layer chromatographic method
reported by Mitchell et al. (7) was quite time-consuming.
Mandava et al. (16) successfully developed a more rapid
procedure for the production of brassins using column
chromatography. The procedure was based on three steps -
1. the extraction of dried rape pollen with ethyl ether to
yield a crude extract, 2. the chromatographic clean-up of
the crude extract on a silica gel column with benzene-
methanol-acetic acid (45:8:4,v:v:v) to yield a biologically
active extract, and 3. the further purification of the
active extract v ia preparative silica gel thin layer
1 chromatography with diisopropyl ether-acetic acid
0
h0 (95:5,v:v) to yield brassins. The other phase of the
c
4. chemistry program continued to be directed towards
7
4 determining the chemical identity of the active
0
1- component(s) of brassins. At this point Mandava and
9
9 Mitchell (17) speculated that brassins were composed of
1
k- fatty acids linked by an ester bond to glucose at carbon 1
b
1/ in the /?-form.
2
10 Prior to his retirement from the USDA in 1974,
10. Mitchell sought increased support to establish field trials
oi: to evaluate the effects of brassins for enhancing yields of
1 | d several crop plants. Because the purification, isolation,
9 and identification of the active component(s) were proving
9
1 to be so very difficult, Mitchell also sought additional
er 4, support for a concerted research effort in these area. The
b ARS administration considered both aspects to be of
m
e importance and plans for a multi-disciplined, multi-
v
No location project were made.
e:
at 1974 to 1980 - NRRC, Peoria, IL; ERRC, Philadelphia, PA;
D
n BARC, Beltsville, MD
o
ati
blic In 1974 plans were submitted by the NRRC, ERRC, and BARC
u outlining the extent and type of input that would be
P
available from each location for extracting large amounts
of brassins from rape pollen and for the chemical isolation
and identification studies. The overall program was
coordinated at Beltsville (G.L. Steffens with assistance
from M. Jacobson for chemical aspects). The pilot plant-
size extraction of rape pollen was conducted at ERRC (M.F.
Kozempel); the large scale chromatographic clean-up of the
crude extract for the production of active brassins was
done at BARC (N. Mandava) ; and the isolation, purification
and identification of the active component(s) of brassins
were undertaken at NRRC (M.D. Grove) and BARC (N. Mandava).
All bean 2nd internode assays required to follow biological
activity during a ll stages of the project were conducted at
BARC (J.F. Worley and D.W. Spaulding). Field studies in
the U.S. and Brazil were also coordinated through BARC
6 BRASSINOSTEROIDS: CHEMISTRY, BIOACTIVITY, AND APPLICATIONS
(G.L. Steffens). A number of other scientists made major
contribution to the program, as will be shown. It was a
multi-phase project and each phase will be discussed
separately although several phases were usually being
conducted simultaneously.
Purification, Isolation, and Identification of Active
Component(s) of Brassins. As indicated, the brassins
complex was found to contain glucosyl esters of fatty acids
(17) which were thought to be the active brassins
constituents. Starting with this information, glucosyl
esters from rape pollen were purified and esters of these
types were synthesized (18, 19, 20) . However, neither the
esters purified from pollen nor those synthesized proved to
have brassins-like biological activity in the bean 2nd
1 internode test. It was therefore concluded that components
0
0 other than glucosyl esters, most likely present in very
h
4.c small amounts, were responsible for the biological activity
47 of brassins.
0
1- Because of the apparent low concentration of the
9
9 active component(s) in the brassins complex, it was
1
k- necessary to plan for the extraction of a large quantity of
b
1/ rape pollen. Honeybee-collected rape pollen was available
2
0 from Canada since pollen is used in specialty-type natural
1
0. food products, for example baked goods. Arrangements were
1
oi: made (not without some difficulty) to obtain a large lot
d (over 1/4 ton ) of rape pollen from Canada for a pilot
91 | plant-scale extraction at ERRC. Earlier work by Mandava et
19 al. (16) was the basis for the pilot plant extraction
4, procedure that was developed and used (21) . Contributors
ber to the project, in addition to M. Kozempel and N. Mandava,
m
e were H. Kenney of ERRC; J.F. Worley, D. Matthees, J.D.
v
o Warthen, Jr., M. Jacobson and G.L. Steffens of BARC; and
N
e: M.D. Grove, NRRC.
at
D
n Pilot Plant Extraction - ERRC. An outline of the
o
ati pilot plant extraction steps is shown in Figures 1 and 2.
ublic oBfa t2c2h7 esk go fp oplollelne)n w(e5r eb aetxcthreasc toefd f45i.r5 st kwgi the adcehi,o nfiozr eda wtaottearl
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in a 227 L kettle to remove simple sugars. The aqueous
slurry was filtered via a Sparkler filter and the filter
cake continuously washed with water until the filtrate was
clear and colorless. The washed pollen (filter cake) was
then freeze-dried, moved back to the extraction kettle and
extracted with 114 L of 2-propanol. The slurry was pumped
to a Sparkler filter, allowed to soak, and the 2-propanol
drained off. The pollen cake was extracted in this manner
7 more times and then it was recycled to the extraction
kettle where it was again extracted with 114 L of 2-
propanol. After filtration, the pollen was washed twice
with 2-propanol and then recycled to the extraction kettle
for a final 2-propanol extraction (a total of eleven 2-
propanol extractions). The 2-propanol was evaporated in a
glass evaporator at <40°C to yield ca. 7.6 L of concentrate