ABA as a Universal Plant Hormone
Yoichi Sakata,Kenji Komatsu,and Daisuke Takezawa
Contents
1Introduction (58)
2ABA in Non-seed Plant Lineages (61)
2.1ABA in Bryophytes (61)
2.2ABA in Ferns and Lycophytes (62)
3Evolution of ABA-Related Genes (64)
3.1ABA Biosynthesis and Metabolism (64)
3.2ABA Transporters (66)
3.3ABA Signaling Core Components (70)
3.4Transcription Factors (71)
4Evolutionarily Conserved Function of ABA (72)
4.1Desiccation Tolerance (72)
4.2Low Temperature Responses (74)
4.3Stomatal Closure (75)
5Role of Cytosolic Calcium and Calcium-Binding Proteins in ABA Signaling (78)
5.1CDPKs (78)
5.2Other CaBPs (79)
5.3The Role of Calcium in Basal Land Plants (83)
6Conclusions and Perspectives (84)
References (86)
Y.Sakata(*)
Department of Bioscience,Tokyo University of Agriculture,1-1-1Sakuragaoka,Setagaya-ku, Tokyo156-8502,Japan
e-mail:sakata@nodai.ac.jp
K.Komatsu
Department of Bioproduction Technology,Junior College of Tokyo University of Agriculture,
1-1-1Sakuragaoka,Setagaya-ku,Tokyo156-8502,Japan
e-mail:k4komats@nodai.ac.jp
D.Takezawa
Graduate School of Science and Engineering and Institute for Environmental Science
and Technology,Saitama University,Saitama338-8570,Japan
e-mail:takezawa@mail.saitama-u.ac.jp
57 U.Lu¨ttge et al.(eds.),Progress in Botany,Progress in Botany75,
DOI10.1007/978-3-642-38797-5_2,?Springer-Verlag Berlin Heidelberg2014
58Y.Sakata et al. Abstract Abscisic acid(ABA)is a sesquiterpene known to regulate environmental stress responses in angiosperms,such as water-loss-induced stomatal closure, development of seed desiccation tolerance during maturation,and salt-, desiccation-,and freezing-stress tolerance of vegetative tissues.An ABA-induced increase in stress tolerance is also reported in other land plant lineages,including nonvascular bryophytes that diverged from vascular plants more than420million years ago.Thus,it is hypothesized that acquisition of sensing and response mechanisms for ABA by land plant ancestors was critical for invasion of and adaptation to land.Because bryophytes are key organisms in plant evolution, clari?cation of their ABA-dependent processes is important for understanding land plant evolutionary adaptation.Based on past and current studies on ABA in non-seed plants and phylogenetic analysis of genome information from various plant species,we discuss the evolution of ABA function and biosynthesis,transport, and signaling network pathways as well as calcium signaling because of its importance in ABA signaling in angiosperms.Future directions of ABA research in the evo-devo?eld are also discussed.
1Introduction
The plant hormone abscisic acid(ABA)can be found in various species across kingdoms,including bacteria,fungi,and animals,as well as plants.The roles or functions of ABA in these non-plant species are largely unknown;however,recent reports suggest a ubiquitous role of the sesquiterpene in the regulation of physiological events in non-plant species(Takezawa et al.2011).Even in the plant kingdom, our knowledge about ABA function largely depends on studies in angiosperms,and a fundamental understanding of ABA activity has just begun to develop for non-angiosperm species.In vascular plants,ABA is produced under water-stress conditions in vegetative tissues and controls stomatal closure and gene expression related to dehydration tolerance(Finkelstein and Rock2002;Rock et al.2010).It also regulates maturation,acquisition of desiccation tolerance,and germination of seeds (Finkelstein et al.2002).In addition,ABA has diverse functions involved in negative control of growth and development such as inhibition of lateral root growth and in?orescence formation(Milborrow1974).Recent progress in molecular studies of ABA function in basal land plants has revealed its ancient origin in water-stress responses in land plants(Fig.1)(Takezawa et al.2011).We can readily note that ABA functions evolved in land plants as a precursor to the acquisition of stomata, vascular system,and seeds that are its target tissues in angiosperms.Thus,understanding the physiological roles as well as the biosynthesis and metabolic and signaling pathways of ABA in non-seed plants will yield insights into how the ubiquitous compound became a plant hormone that is essential for the water-stress response in land plants.
Bryophytes such as mosses and liverworts are nonvascular plants that?rst emerged 480million years ago and are widespread across the world from tropical rain forests to regions with harsh environmental conditions such as the desert or Antarctic.
Because they lack water transport and retention tissues,bryophytes engage in a continual equilibrium with the water potentials of the atmosphere (à100MPa at 28 C and 50%relative humidity),a level most modern-day ?owering plants could not tolerate.But when watered,they can equilibrate rapidly with surrounding water potential and are fully hydrated without signi?cant damage.This type of desiccation tolerance is likely an ancestral trait of land plants that has been lost from the vegetative tissues of vascular plants during evolution but “re-evolved”in the Selaginellas and the Leptosporangiate ferns (Oliver et al.2000,2005).The mechanisms underlying desic-cation tolerance include (1)limiting damage of cellular components caused by severe dehydration stress;(2)maintaining physiological integrity during desiccation;and (3)provoking the cellular repair mechanism upon rehydration (Bewley 1978).
It is critical to ?nd non-angiosperm plants for which tools are available for gene discovery and functional analysis.The moss Physcomitrella patens is one of the few such plants (Quatrano et al.2007).After ?ndings indicating a high frequency of homologous recombination that enables gene targeting (Schaefer and Zryd 1997),this plant has emerged as a model system for analysis of many aspects of plant biology.In 2008,an assembled genome of P.patens (487Mbp)was released from the Joint Genome Institute in the USA (JGI)(Rensing et al.2008).The predicted gene number is 32,272which is comparable to ?ndings in angiosperms,and generation of a linkage map with molecular markers is in progress (Kamisugi et al.2008),which will be a resource for forward genetics.RNA interference (RNAi)(Bezanilla et al.2003)and arti?cial microRNAs (Khraiwesh et al.2008)can be used to downregulate gene expression,an alternative to targeted gene disruption for analysis of gene families in this organism.Taking advantage of this wealth of tools for comparative and functional analysis,molecular dissection of
Hornworts Liverworts Mosses Lycophytes Ferns Gymnospems Angiosperms
500400450300
(mya)
350Ancestral green algae
Presence Effect
ABA
Stoma
Seed Desiccation tolerance
Fig.1ABA and land plant evolution.The cladogram shows the evolutionary relationship of land plant groups.Relative effect (blue )and endogenous level (red )of ABA in each plant groups are shown by dot size.ABA effectiveness in seed development,stomatal regulation,and desiccation tolerance is indicated by the bar chart .Color strength shows effectiveness
ABA as a Universal Plant Hormone
59
ABA signaling pathways as well as the biosynthesis pathway in basal land plants is ongoing.For example,recent studies on P.patens have revealed developmental and physiological aspects of ABA response in bryophytes (Fig.2).Gene knockout experiments indicated that desiccation tolerance in the moss involves ABA and the plant-speci?c transcription factor ABA INSENSITIVE3(ABI3),which are essential for seed desiccation tolerance of angiosperms (Marella et al.2006;Khandelwal et al.2010),suggesting an evolutionarily conserved mechanism for cellular protection from https://www.doczj.com/doc/9c2775165.html,parative analyses of the common toolkit for ABA functions in land plants will further extend our understanding of the evolution of the signaling machinery that enabled plants to conquer the land.
In this review,we focus on the role of ABA in land plants outside of seed plants including bryophytes,lycophytes,and ferns,and recent progress in the elucidation of an ABA-core toolkit in the model moss P.patens and an emerging new model the liverwort Marchantia polymorpha .We further discuss the evolution of ABA function,ABA metabolism,and its signaling pathway in relation to calcium signaling that must be related to the innovation of water use ef?ciency during the evolution of land plants.
gametophore (n)
chloronema
caulonema
brood cell
protomema (n)
bud formation
sporophyte (2n)formation
sex o rgans formation
rhizoid
rhizoid formation (auxin)
gametophore formation
ABA
spore
germination
spore (n)
auxin cytokinin
ABA
auxin cytokinin
P. patens
life cycle
a
b
d
e
f
h c
g
i
j k
l
Fig.2The life cycle of the moss Physcomitrella patens.(a )Spores,(b )spore germination and generating primary chloronemata,(c )chloronema cells,(d )caulonema cells,(e )caulonema differentiation is regulated by auxin and cytokinin,(f )brood cell formation is regulated by ABA,(g )young bud,(h )auxin and cytokinin induce bud formation,and ABA represses the formation,(i )gametophore (leafy shoot)and rhizoid (rootlike tissue),(j )auxin induces rhizoid formation,(k )a short day and a cold treatment (15 C)induce sex organ formation,(k )after fertilization,the egg cell develops into a sporophyte (diploid)and within its meiosis occurs leading to spore formation.The cycle can be achieved under laboratory conditions in less than 3months
60
Y.Sakata et al.
ABA as a Universal Plant Hormone61 2ABA in Non-seed Plant Lineages
2.1ABA in Bryophytes
Detection of endogenous ABA in various species of bryophytes by enzyme-linked immunosorbent assays(ELISA)using monoclonal antibodies has been reported. Analysis of endogenous ABA in the desiccation-tolerant liverwort Exormotheca holstii has revealed that desiccation of its thalli resulted in a drastic increase in ABA content in comparison with the hydrated thalli(Hellwege et al.1992). Further analysis indicated that various liverwort species belonging to Marchantiales accumulate endogenous ABA with a large variability in the content ranging from 2pmol gà1FW to30nmol gà1FW(Hartung and Gimmler1994).In hornworts (Anthoceros species),ABA was detected not only in the gametophyte but also in the sporophyte,and stress-induced ABA synthesis was observed(Hartung et al.1987). In the moss Funaria hygrometrica,ABA accumulated during the slow drying process associated with development of desiccation tolerance(Werner et al. 1991).However,these ABA studies require cautious interpretation.Minami et al. (2005)described that the amount of ABA could not be determined precisely by ELISA at least in P.patens,probably because of cross-reactivity of the anti-ABA antibody used in the assay with other compounds in crude extracts.Direct mea-surement of endogenous ABA has been conducted in only limited species of bryophytes.Li et al.(1994)estimated that cultured thalli of M.polymorpha contain 4–16ng of ABA gà1FW by direct measurement by GC-EIMS and GC-NCIMS.In mosses,endogenous ABA has been detected by GC-MS in protonema cells of P.patens,and it was found that the cells accumulated more ABA in response to hyperosmotic stress(Minami et al.2005).
Exogenous application of ABA affects growth and development as well as stress tolerance in bryophytes.In mosses,exogenous ABA inhibits growth of protonema and gametophore(Menon and Lal1974;Lehnert and Bopp1983;Chopra and Kapur1989).ABA also promotes formation of gemma-like structures or spherical “brood cells,”which are forms of vegetative reproduction(Chopra and Kapur1989; Goode et al.1993;Schnepf and Reinhard1997).Another well-documented effect of ABA on protonemata is inhibition of gametophore formation.ABA inhibits caulonema’s differentiation to gametophore by counteracting with cytokinin that promotes bud formation(Valadon and Mummery1971;Chopra and Kapur1989; Christianson2000).There are also reports indicating that ABA inhibits formation of gametangia for sexual reproduction(Bhatla and Chopra1981;Chopra and Mehta1987).In comparison to the studies in mosses,there are a relatively limited number of reports describing the effects of ABA on growth and development of liverworts.ABA inhibits growth of gemmae and thalli(Schwabe and Valio1970) and formation of gametangia in Marchantiales liverworts(Kumra and Chopra 1986).ABA might play a role in heterophyllous switch of liverworts.Hellwege et al.(1992)reported that exogenous ABA converted thalli of aquatic liverworts Riccia?uitans and Ricciocarpus natans into the land forms.
62Y.Sakata et al.
In addition to above studies,effects of exogenous ABA on tolerance to desiccation and freezing stress in bryophytes are well described.Application of ABA to cultured protonemata of F.hygrometrica induces tolerance to rapid drying(Werner et al. 1991).Pretreatment with ABA of gametophores of Atrichum androgynum reduces membrane ion leakage caused by desiccation stress(Beckett1999).ABA appears to prevent reductions in photosynthesis and non-photochemical quenching caused by desiccation in A.androgynum(Mayaba et al.2001).Effects of ABA on freezing tolerance have been also described.ABA pretreatment of protonemata with cryoprotectants such as proline and sucrose effectively enhanced survival after cryopreservation of moss species(Christianson1998;Burch and Wilkinson2002). In P.patens protonemata,ABA treatment for one day increases freezing tolerance fromà2 C toà10 C(Minami et al.2003).The protonemata accumulate soluble sugars as compatible solutes as well as a number of LEA-like proteins and stress-associated transcripts by ABA treatment(Nagao et al.2005,2006).
Exogenous ABA also induces freezing and desiccation tolerance of some liverworts.Pence(1998)showed that ABA pretreatment enhanced survivals after cryopreservation of liverwort species,although the magnitude of the effects of ABA varied among species.ABA effectively increased desiccation tolerance of Riccia?uitans while the effect was little on Plagiochila sp.and M.polymorpha (Pence et al.2005).The ABA-induced desiccation tolerance appeared to be associated with increased accumulation of total soluble carbohydrates(Pence et al. 2005),though pro?les of proteins and gene expression have not been described. These results suggested that effects of ABA on liverworts may vary substantially among different species,but it certainly alters development and desiccation toler-ance of some species such as R.?uitans.Most of the studies on ABA effects are those on Marchantiales liverworts and very little is known about its effects on other liverwort groups including Jungermanniales.
2.2ABA in Ferns and Lycophytes
Radioimmunoassays for detection of ABA indicated its distribution in pteridophytes (Weiler1979).Endogenous ABA has been chemically identi?ed in different species of pteridophytes:protonema of Anemia phyllitidis(Cheng and Schraudolf1974), spores of Lygodium japonicum(Yamane et al.1980),sporophytes of the tree ferns Cibotium glaucum and Dicksonia antarctica(Yamane et al.1988),and sporophytes of an aquatic fern Marsilea drummondii(Pilate et al.1989).Recently,detection by more accurate physicochemical methods(GC-MS/MS,GC-SIM,and UPLC-MS/ MS)of water-stress-induced foliar ABA levels in ferns(Pteridium esculentum and D.antarctica)and a lycophyte(Selaginella kraussiana)has been reported(Brodribb and McAdam2011;McAdam and Brodribb2012).These analyses revealed that foliar ABA levels of ferns and lycophytes are quite low(about10ng gà1FW or below)in unstressed control conditions but are markedly increased(about 200–600ng gà1FW)when exposed to water stress,as observed in seed plants.
ABA as a Universal Plant Hormone63 Different physiological roles of ABA in growth and environmental responses have been reported.
ABA has a negative effect on growth of gametophyte tissues in ferns.General retardation of growth by ABA of the gametophyte of L.japonicum that shows characteristic growth behavior under red and blue light has been reported by Swami and Raghavan(1980).ABA inhibits protonemal elongation,but not spore germination,in Mohria caffrorum(Chia and Raghavan1982).Hickok(1983) showed that ABA stimulates rhizoid production of the Ceratopteris gametophyte at concentrations of10à6–10à5M but is inhibitory at higher concentrations.ABA has been shown to regulate sex determination in Ceratopteris.The fern Ceratopteris produces either hermaphroditic or male gametophytes,and this developmental fate is determined by the antheridiogen(possibly a gibberellin-like substance) produced by the hermaphroditic gametophytes.Hickok(1983)showed that ABA inhibits antheridiogen-induced formation of male gametophytes.Analysis of ABA-insensitive mutants indicated that these plants develop as males in the presence of antheridiogen and ABA(Warne and Hickok1991).
ABA also has been shown to control heterophyllous switch in aquatic fern plants.The sporophyte of Marsilea quadrifolia produces different types of leaves above and below the water level.It has been shown that its submerged type shoots are modi?ed by ABA,and the development of aerial-type leaves is induced(Liu 1984).Here ABA promoted growth of both leaves and roots but inhibited growth in the internodes.ABA-induced developmental changes returned to the default mode when ABA was removed.A number of ABA-regulated genes,designated ABRH for ABA-responsive heterophylly,have been identi?ed from the shoot apex tissues of M.quadrifolia(Hsu et al.2001).Of interest,unnatural R-(à)-ABA has a greater effect on induction of heterophylly and the ABRH gene expression than natural S-(+)-ABA,which is possibly due to slow70-hydroxylation of R-(à)-ABA for its metabolism(Lin et al.2005).The heterophyllous switch between aquatic and land forms by ABA has been observed in a wide range of plant species from angiosperms to liverworts(Anderson1978;Hellwege et al.1992).Whether or not the mechanisms for these morphogenetic responses by ABA are conserved among different classes of land plants of distantly related taxa is yet to be determined.
The role of ABA in desiccation tolerance of both ferns and lycophytes has been reported.Detached fronds of the desiccation-tolerant fern Polypodium virginianum survive slow drying but are severely damaged by drying with silica gel due to rapid water loss.When the fronds are treated with ABA,the amount of water lost is reduced,resulting in better survival after silica gel drying(Reynolds and Bewley 1993a).This process accompanied de novo synthesis of speci?c proteins(Reynolds and Bewley1993b).The effect of exogenous ABA on survival after open drying followed by liquid nitrogen storage for cryopreservation of gametophyte tissues of six fern species was examined,and preculture with10μM ABA for one week increased survival of the tissues of all species(Pence2000).Liu et al.reported that ABA functions in desiccation tolerance in the lycophyte.Dehydration treatment of the sporophyte of Selaginella tamariscina causes a threefold increase in endogenous
64Y.Sakata et al. ABA content,associated with upregulation of expression of genes involved in ABA signaling and cellular protection(Liu et al.2008).
3Evolution of ABA-Related Genes
With the advance of sequencing technology of DNA,genomic information of various plant species is now available(Phytozome;https://www.doczj.com/doc/9c2775165.html,). Here we summarize the comparison of numbers of ABA-related genes that are involved in ABA biosynthesis and catabolism and ABA transport and the signaling among plant lineages from green algae to angiosperms(Table1)and describe insights into the evolution of ABA-related genes in plants.We note that the sequenced plant species are still biased and genomic information from representatives of gymnosperms,ferns,hornworts,and liverworts will be required to complete an evolutionary comparison of ABA-related genes.
3.1ABA Biosynthesis and Metabolism
The rates of ABA biosynthesis and catabolism are critical to determining the accumulation of ABA as well as the strength of the response.Although a variety of organisms synthesize ABA,the carotenoid pathway in angiosperms is the only de?ned pathway for ABA biosynthesis(Nambara and Marion-Poll2005).The Arabidopsis ABA1encodes zeaxanthin epoxidase(ZEP),which catalyzes the initi-ation step of the carotenoid pathway.The cyanobacterium Synechocystis genome lacks genes for ABA1(Yoshida2005).Our phylogenetic analysis also showed no ZEP in the primitive red alga Cyanidioschyzon merolae(Takezawa et al.2011)but identi?ed one ZEP ortholog in C.reinhardtii(Table1).The genome of the basal land plant P.patens contains a gene set for the carotenoid pathway except for ABA2,suggesting that the carotenoid pathway was completed in ancestral land plants.ABA2appeared only in angiosperms,suggesting the presence of alternative enzymatic pathway to convert xanthoxin to ABA https://www.doczj.com/doc/9c2775165.html,nd plants from bryophytes to angiosperms increase ABA accumulation upon water stresses (Takezawa et al.2011;McAdam and Brodribb2012).NCED(9-cis-epoxy-carotenoid dioxygenase)catalyzes the rate-limiting step of water-stress-induced ABA biosynthesis in angiosperms(Nambara and Marion-Poll2005),and NCED expression is upregulated by water stress in angiosperms(Xiong and Zhu2003). The moss P.patens is currently the most basal fully sequenced land plant and accumulates ABA upon osmostress(Minami et al.2005).The P.patens genome encodes two NCED genes(Table1)with expression upregulated by dehydration, salinity,and exogenous ABA(Richardt et al.2010),as observed in vascular plants. These data suggest that water-stress-controlled biosynthesis of ABA was established in the last common ancestor of land plants.Disruption of these genes for the carotenoid pathway of ABA biosynthesis in the basal land plant P.patens
T a b l e 1C o m p a r i s o n o f n u m b e r o f a b s c i s i c a c i d (A B A )-r e l a t e d g e n e s a m o n g p l a n t s
F u n c t i o n
G e n e G r e e n a l g a e B r y o p h y t e s L y c o p h y t e s A n g i o s p e r m s
C .r e i n h a r d t i i P .p a t e n s S .m o e l l e n d o r f ?i O .s a t i v a A .t h a l i a n a A B A m e t a b o l i s m
A B A 1/Z E P 11111A B A 401111N C E D s 02135A B A 200011A A O 300001A B A 311101B G 100001C Y P 707A s 00234A B A t r a n s p o r t A B C G 2500011A B C G 40000101A I T 00014A B A s i g n a l i n g P Y R /P Y L /R C A R s 0451114G r o u p A P P 2C 023109S u b c l a s s I I I S n R K 204233A B I 303311A B I 400011A B I 502457S L A C 104395C a 2+-d e p e n d e n t f a c t o r C I P K /S n R K 30742925C D P K 2030103134C B L 2431010C a M /C M L 929263757T h e A r a b i d o p s i s A B A 1,A B A 4,N C E D 3,A B A 2,A A O 3,A B A 3,B G 1,C Y P 707A 3,A B C G 25,A B C G 40,A I T 1,P Y R ,A B I 1,S R K 2E ,A B I 3,A B I 4,A B I 5,S L A C 1,C I P K s ,C D P K s ,C B L s ,a n d C M L s a m i n o a c i d s e q u e n c e s w e r e u s e d f o r a B L A S T P s e a r c h t o d e t e r m i n e t h e p u t a t i v e o r t h o l o g s i n ?v e s p e c i e s ,a n d t h e n e i g h b o r -j o i n i n g (N J )t r e e s w e r e d r a w n u s i n g M E G A 5.0.T h e n u m b e r o f p o t e n t i a l o r t h o l o g s w a s c o u n t e d w i t h t h e N J -t r e e s .T h e d a t a b a s e u s e d w a s P h y t o z o m e v 8.0(h t t p ://w w w .p h y t o z o m e .n e t )
ABA as a Universal Plant Hormone
65
66Y.Sakata et al. via gene targeting will uncover the signi?cance of the carotenoid pathway for ABA biosynthesis as well as the physiological roles in nonvascular plants.
Several genes involved in ABA catabolism have been identi?ed in Arabidopsis. ABA hydroxylation at the80position to give phaseic acid is the main irreversible catabolic pathway in angiosperms(Nambara and Marion-Poll2005).The metabolites 70-and90-hydroxy ABA are also reported in some angiosperm species(Zhou et al. 2004).To date,only CYP707A genes for80-hydroxylation of ABA have been reported in Arabidopsis(Kushiro et al.2004),and these genes are involved in drought tolerance (Umezawa et al.2006)and seed dormancy as well as germination(Okamoto et al. 2006).Another catabolic pathway is conjugation of ABA to glucose-ester(GE),which is subsequently sequestered into vacuoles(Boyer and Zeevaart1982;Bray and Zeevaart1985).ABA-GE is reversibly converted to ABA by BETA GLUCOSIDASE (BG)in Arabidopsis.To date,two Arabidopsis BG genes,endoplasmic reticulum-localized AtBG1and vacuole-localized AtBG2,have been reported to catalyze ABA-GE hydrolysis(Lee et al.2006;Xu et al.2012).AtBG1disruption results in reduced ABA accumulation in seeds(about40%of WT)and in water-stressed leaves (about80%of WT).The atbg1plants show not only ABA-de?cient phenotypes but also a dwarf phenotype with yellow leaves that can be rescued by exogenous ABA (Lee et al.2006).These data suggest roles for released ABA in the stress response as well as in normal development of Arabidopsis.The authors suggested that this reversible pathway is involved in water-stress-induced rapid ABA accumulation that does not require gene activation.However,a relatively small impact of loss of AtBG1on ABA accumulation in water-stressed leaves compared to the other phenotypes may suggest roles for AtBG1beyond ABA release.We also note that no direct evidence of ABA production is provided for the atbg2plants that exhibit phenotypes similar to those of the atbg1plants(Xu et al.2012).
An unexpected result was that P.patens does not possess genes for any of these enzymes for ABA catabolism.CYP707A genes are present only in vascular plants,and BG1is present only in angiosperms(Table1and Fig.3).Our preliminary experiment to detect ABA catabolites showed that protonemata of P.patens do not accumulate either ABA-GE or80-hydroxylation catabolites.Instead,we detected only90-hydroxylation catabolites(neoPA)(Sakata et al.unpublished results).P.patens actively exports synthesized ABA to the extracellular environment(Minami et al.2005).The extra-cellular export system,rather than inactivation and sequestration,is likely to function as the major system to reduce intercellular ABA level in bryophytes.Recently,Okamoto et al.(2011)reported that ABA90-hydroxylation is catalyzed by CYP707A as a side reaction in Arabidopsis.Bryophytes may possess other CYPs that catalyze90-hydrox-ylation of ABA as the main reaction.
3.2ABA Transporters
ABA of angiosperms has been considered as a root-derived signaling molecule that induces physiological changes in shoots in response to dry soil conditions.In fact, expression of Arabidopsis AAO3,AtABA2,and AtANCED3genes is observed in
vascular tissues (parenchyma cells)in response to water stress (Endo et al.2008;Koiwai et al.2004;Seo and Koshiba 2011),indicating that ABA produced in vascular tissues must be delivered to the cells such as guard cells that require water-stress responses.The molecular basis of ABA transportation was only recently clari?ed.Two independent research groups simultaneously made the breakthrough for ABA transporters using Arabidopsis.Kang et al.(2010)reported that a
At Arabidopsis thaliana Os Oryza sativa
Sm Selaginella moellendorffii Pp Physcomitrella patens
gene names
CYP707A
(cytochrome P450, ABA 8'-hydroxylase)
At3G18080
Os01g32364Os03g49600Sm149851
Os12g23170Os01g67220Pp1s76 8V6Sm268319Os06g46940Os10g17650Os03g11420Sm163822Sm75234Sm163827Sm268527Sm76384
Sm76748Sm228612
Sm127964Sm153534
Pp1s137 79V6Pp1s114 133V6Pp1s9 78V6Os04g39840Os04g39864Os04g39880Os04g39900Os06g21570
Os09g31430
Os08g39870Os08g39860
At2G44460At2G44470At3G60140At5G24550
At5G24540
At2G32860At2G44490At3G60120
AtBG1
At3G21370At1G75940At5G28510At3G09260At1G66270At1G66280
100
10084
100100
58
89
87
100
100
100
99
91
100
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100
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99
100
98100
6056
48100
6392100
82
97
83
98
5887
50
46
9663
28
100
23
21
70.05
0.1
BG1
(β-glucosidase)
b
Fig.3Phylogenetic relationship among orthologs of the CYP707A family and BG1glucosidase.The phylogenetic tree of CYP707A family (a )and BG1glucosidases (b )was generated by MEGA5.0by using the neighbor-joining method.Genes of A.thaliana,O.sativa,S.moellendorf?i ,and P.patens are indicated by red ,blue ,green ,and black ,respectively.Previously reported Arabidopsis ABA 80-hydroxylase CYP707A family and ABA glucosidase BG1are indicated by arrowheads
ABA as a Universal Plant Hormone 67
68Y.Sakata et al. pleiotropic drug resistance transporter(PDR)-type ABC transporter PDR12 (AtPDR12/AtABCG40)mediates uptake of ABA into plant cells,and expression of AtPDR12/AtABCG40in yeast cells and BY2cells increases ABA uptake. Furthermore,protoplasts derived from atabcg40knockout mutant plants showed reduced uptake of ABA.AtPDR12/AtABCG40is membrane localized and expressed ubiquitously with higher expression in guard cells.The atabcg40plants show a delay in gene expression in response to ABA application and are impaired in stress tolerance.Kuromori et al.(2010)isolated an Arabidopsis mutant line with an ABA-sensitive phenotype in the germination and seedling stages from activator (Ac)/dissociation(Ds)transposon-tagged mutant collection.The Ds element was inserted into the At1g71960(AtABCG25/AtWBC26)gene.Expression of AtABCG25 was also ubiquitous with the most obvious expression in the hypocotyls,roots,and vascular veins of leaves and enhanced by ABA treatment.Subcellular localization of AtABCG25was observed at the plasma membrane.ATP-dependent ABA transport activity was observed in membrane vesicles derived from AtABCG25-expressing insect cells,suggesting that AtABCG25functions as an ABA exporter.Kuromori et al.(2011)also suggested the involvement of AtABCG22—which is closely related to AtABCG25in the phylogenic tree—in ABA function in guard cells, although direct evidence for ABA transport activity is lacking.More recently, Kanno et al.(2012)identi?ed a novel ABA transporter using an elegant experimental design with Arabidopsis cDNAs capable of inducing interactions between PYR/ PYL/RCAR and PP2C in yeasts under low ABA concentrations.The isolated gene, designated ABA-IMPORTING TRANSPORTER(AIT),encoded the low-af?nity nitrate transporter NRT1.2,belonging to the NRT1/PTR transporter family,which consists of53related sequences(Tsay et al.2007).AIT1shows a higher af?nity for the natural(+)-ABA than for the synthetic(à)-ABA as its substrate,and is insensitive to an ABA agonist,pyrabactin,indicating speci?c recognition of ABA structure.We note that AtABCG40and AtABCG25had no effect in this assay.AIT1 was localized at the plasma membrane of Arabidopsis cells,and the promoter activity was observed in imbibed seeds and in vascular tissues in cotyledons,true leaves,hypocotyls,roots,and in?orescence stems after germination.The ait1/nrt1.2 plants showed a lower surface temperature in the in?orescence stems than those of the WT due to the excess water loss from open stomata.The?nding of an ABA transporter that also transports nitrate partly explains the cross talk between ABA signals and nitrate(Matakiadis et al.2009).
Of interest,none of these ABA transporters can be found in either the genome of the moss P.patens or that of the basal vascular plant S.moellendorf?i(Table1and Fig.4).We know that P.patens exports intercellular cytosolic ABA outside of cells (Minami et al.2005)and that membrane proteins likely drive this action. Arabidopsis ABA transporters described here might evolve after emergence of seed plants,and there must be other ABA transporters that are yet to be identi?ed.
At Arabidopsis thaliana Os Oryza sativa
Sm Selaginella moellendorffii Pp Physcomitrella patens
gene names
0.1
AIT
(NRT/PTR family transporter)
AtABCG25
(WBC type ABC transporter)
0.05
PDR12/AtABCG40
(PDR type ABC transporter)
a
Fig.4Phylogenetic relationship among orthologs of ABA transporters.The phylogenetic tree of PDR12/AtABCG40(a ),AtABCG25(b ),and AITs (c )was generated by MEGA5.0by using the neighbor-joining method.Genes of A.thaliana,O.sativa,S.moellendorf?i ,and P.patens are indicated by red ,blue ,green ,and black ,respectively.Previously reported ABA transporters,PDR12/AtABCG40,AtABCG25,and AITs,are indicated by arrowheads
ABA as a Universal Plant Hormone 69
70Y.Sakata et al.
3.3ABA Signaling Core Components
Compared to enzymes for ABA metabolism and transporters,ABA signaling molecules are well conserved among land plants.Studies in Arabidopsis identi?ed several candidates of ABA receptors including membrane-integrated G-protein-coupled receptors(GPCRs)(Liu et al.2007),GPCR-type G proteins(Pandey et al.2009),and the chloroplast-localized Mg chelatase H subunit(Shen et al.2006);however,their functions as ABA receptors have been questioned or disproven(Risk et al.2009; Tsuzuki et al.2011).Only the cytosolic pyrabactin resistance1/pyrabactin resistance 1-like/regulatory component of ABA receptor(PYR/PYL/RCAR)(Ma et al.2009; Park et al.2009)establishes a robust link to central components of the ABA signaling pathway:plant-speci?c SNF-related protein kinases(SnRK2s)as positive regulators and ABI1-related type-2C protein phosphatases(PP2Cs)as negative regulators (Takezawa et al.2011;Umezawa et al.2010).The three ABA-core components are considered to form an ABA receptor complex to trigger the ABA signaling cascade. ABA-triggered phosphorylation of the transcription factor(Fujii and Zhu2009)and S-type anion channel activation(Brandt et al.2012)by the ABA-core components have been reconstructed in vitro.Moreover,an Arabidopsis sextuple mutant of PYR/PYL/ RCAR genes exhibits extreme ABA insensitivity in almost all known ABA-related responses,as is expected from their roles as ABA receptors(Gonzalez-Guzman et al. 2012).These results indicate that the cytosolic ABA receptors play a major role in sensing the hormone in Arabidopsis.The ABA-core components described here are well conserved only in land plants,although their functions outside angiosperms are yet to be elucidated.
Recent studies on model bryophytes indicate that group A PP2C,a member of the ABA receptor core components,plays a role in ABA responses in basal land plants.Komatsu et al.(2009)reported a disruption of one of two PpABI1genes (PpABI1A and PpABI1B)encoding Group A PP2Cs in P.patens.Single disruption of PpABI1A by gene targeting causes an ABA-hypersensitive phenotype such as increased stress tolerance and enhanced expression of ABA-inducible genes, suggesting that PpABI1also functions as a negative regulator of ABA signaling in P.patens(Komatsu et al.2009).More recently,we carried out homology search for signaling molecules for ABA responses in M.polymorpha and isolated the MpABI1gene that encodes an ABI1-like PP2C(Tougane et al.2010).To analyze functions of MpABI1,a transient assay system by particle bombardment of cultured cells of M.polymorpha was established and used for analysis of ABA-induced gene expression.In this assay,the cells bombarded with the Em-GUS construct showed ABA-induced GUS expression,and co-expression of the MpABI1with Em-GUS resulted in suppression of the ABA-induced GUS expression.Furthermore,trans-genic P.patens plants stably expressing MpABI1showed a reduced sensitivity to osmotic and freezing stress and did not undergo typical morphological changes by ABA treatment(Tougane et al.2010).These results indicated that MpABI1 functions as a negative regulator of ABA signaling.
ABA as a Universal Plant Hormone71 3.4Transcription Factors
To date,many transcription factors have been described that are involved in ABA responses of angiosperms.Among them,ABI3(Giraudat et al.1992)and ABI5 (Group A bZIP)(Finkelstein and Lynch2000)isolated from the analysis of Arabidopsis ABA-insensitive(abi)mutants(Koornneef et al.1984)are considered to be central to conducting ABA signaling during seed maturation and germination. Of interest,the two transcription factors arose co-instantaneously with other ABA-related genes(Table1).The shared feature of the transcription factors is that they target ABA-responsive elements(ABREs),with ACGT-core sequences present in the promoters of many ABA-responsive genes of angiosperms(Marcotte et al.1989; Carles et al.2002;Hobo et al.1999;Vasil et al.1995;Casaretto and Ho2003). Knight et al.(1995)found that a wheat ABA-inducible Em gene promoter is activated by exogenous ABA in the moss P.patens,demonstrating evolutionarily conserved machinery from ABA perception to gene activation between P.patens and angio-sperms.ABI3,also known as VP1in maize,is an indispensable transcription factor in concert with ABA that regulates maturation and desiccation tolerance of seeds (Giraudat et al.1992;McCarty et al.1989).It was unexpected that ABI3/VP1, known as a seed-speci?c transcription factor,originated in the ancestral land plants. Marella et al.(2006)?rst reported the function of ABI3from the moss P.patens (PpABI3).PpABI3can activate the Em promoter in both P.patens and barley alleurone cells,and partially complements the phenotypes of Arabidopsis abi3-6 mutant plants.These?ndings indicate an evolutionarily conserved function of ABI3 in land plants.Sakata et al.(2010)performed comparative analysis of Arabidopsis ABI3and PpABI3regarding the activation of the Em promoter in P.patens.PpABI3 showed more dependency on the other cis-element RY sequence,to which the conserved B3domain of ABI3binds(Suzuki et al.1997),while Arabidopsis ABI3 activated the Em promoter either in an ABRE-or RY sequence-dependent manner. The difference might be attributed to less-conserved N-terminal regions(B1and B2) of PpABI3,which are considered to be involved in interaction with other transcription factors(Nakamura et al.2001;Zhang et al.2005).These?ndings suggested that B3 domain-dependent transcriptional regulation of ABI3is evolutionarily conserved between the moss and angiosperms,whereas angiosperm ABI3has evolved to activate ABA-inducible promoters independently from the B3DNA binding domain. Khandelwal et al.(2010)showed that expression of most of the ABA-inducible genes examined was not affected in abi3null P.patens plants,suggesting that ABI3 is not an essential component for ABRE-mediated gene expression in P.patens,as observed in the ABA response of vegetative tissues of angiosperms.
ABI5belongs to Group A bZIP,which has13genes in Arabidopsis(Jakoby et al. 2002).These bZIPs can be further divided into two subgroups based on the conserved N-terminal domains.Nine bZIPs,including ABI5and AREB/ABFs (ABA-RESPONSIVE ELEMENT BINDING)/(ABA-RESPONSIVE ELEMENT BINDING FACTOR),contain three N-terminal conserved regions termed C1, C2,and C3,and one C-terminal conserved region designated as C4,whereas the
72Y.Sakata et al. other four bZIPs lack the C1domain(Bensmihen et al.2002).The former group (ABI5/AREB/ABF family)is characterized by being involved in ABA signaling in seed development and germination(ABI5,EEL,DPBF2/AtbZIP67,DPBF4,and AREB3)or vegetative tissues(AREB1/ABF2,AREB2/ABF4,ABF1,and ABF3) (Bensmihen et al.2005;Finkelstein et al.2005;Fujita et al.2005;Yoshida et al. 2010)through binding to the ABRE of the ABA-regulated genes via the bZIP DNA binding domain(Choi et al.2000;Carles et al.2002;Uno et al.2000).In contrast to ABI3,Group A bZip has been reported to be absent in the P.patens genome and suggested to have?rst appeared in the most recent common ancestor of spermatophytes;thus it may be related to seed formation(Correa et al.2008). However,our phylogenetic analysis identi?ed two genes that encode putative Group A bZIPs(Table1).This fact suggests that the origin of the Group A bZIP is older than proposed.Functional analysis of these genes in P.patens will shed light on elucidation of the core component of the toolkit for ABA response in land plants.
It is also known that different types of bZIP transcription factors recognize the ACGT-containing sequences found in seed-speci?c gene promoters.Opaque2(O2), a Group C bZIP in maize,binds to TCCACGTAGA in the22-kDaα-zein promoter (Schmidt et al.1992).Orthologous genes from wheat(Triticum aestivum)and barley(Hordeum vulgare)are also reported to play the same roles as O2in their corresponding species(Albani et al.1997;On?ate et al.1999).The Group C bZIP is evolutionarily conserved among land plants(Correa et al.2008),the Arabidopsis genome encodes four Group C bZIP genes,and bZIP10and bZIP25have been characterized in association with ABI3.In contrast to maize O2,bZIP10and bZIP25are ubiquitously expressed in Arabidopsis tissues(Lara et al.2003), suggesting an acquired broader range of function outside seeds.These two bZIP proteins physically interact with ABI3in yeast cells,and co-expression of AtbZIP10/25with ABI3results in a remarkable increase in transactivation of the At2S1promoter(Lara et al.2003).A recent study has indicated that hetero-dimerization of AtbZIP10/25with another class of bZIP protein(Group S)is also involved in the ABI3-mediated regulation of seed maturation-speci?c genes in Arabidopsis(Alonso et al.2009).Richardt et al.(2010)reported in their microarray analysis that two bZIP genes belonging to Group E and G are ABA inducible in P.patens.These observations suggest the possibility for the involvement of other classes of bZIPs in the regulation of ABA responses in basal land plants.
4Evolutionarily Conserved Function of ABA
4.1Desiccation Tolerance
The de?nition of desiccation tolerance(DT)is referred as the ability to survive drying to the absolute water contents less than about0.1g H2O gà1dry matter or to a water potential of less than aboutà100MPa(Farrant2010).Several moss species
ABA as a Universal Plant Hormone73 such as Tortula caninervis are known to survive more thanà540MPa(Oliver et al.
2005).Phylogenetic analyses have suggested that DT in vegetative tissue,as
expressed in bryophytes,was lost in the vegetative body of vascular plants during
the evolution;instead,reproductive organs such as seeds have acquired the trait
(Rensing et al.2008).To survive severe water de?cits,plant cells must maintain
physiological integrity during dehydration and repair mechanisms must exist that
function upon rehydration(Bewley1979).Some moss species such as T.ruralis
show constitutive DT without any preconditioning.T.ruralis constitutively
accumulates soluble sugars and late embryogenesis abundant proteins(LEAs),
both of which are suggested to play a major role in protecting cellular integrity
upon desiccation(Charron and Quatrano2009).The desiccated T.ruralis produces
a set of mRNAs as well as polypeptides whose synthesis is unique to the rehydrated
state(Oliver1991;Scott and Oliver1994),suggesting that changes in gene expres-
sion are also important for cellular repair mechanisms upon the rehydration phase.
Desiccation tolerance of seeds in angiosperms strongly associates with ABA
(Finkelstein et al.2002).ABA is also implicated in regulation of DT in bryophytes.
In contrast to the desiccation-tolerant mosses,several moss species such as
P.patens and Funaria hygrometrica are drought tolerant but not tolerant to rapid
drying.Slow desiccation treatment of F.hygrometrica leads to loss of almost all of
their water after24h with an induced level of endogenous ABA of about sixfold
and recovery after rehydration.Application of exogenous ABA at greater than
10μM confers tolerance to rapid drying,and exogenous ABA has no effect on
the rate of water loss(Werner et al.1991).These data suggest that the DT of
F.hygrometrica is not constitutive but inductive and that ABA plays a key role in
the induction.This ABA-induced desiccation tolerance has been further analyzed at
the molecular level using the model moss P.patens.Without preconditioning
P.patens does not survive water potentials lower than aboutà13MPa;however, 50μM ABA treatment prior to desiccation increases survival against at least à273MPa(Koster et al.2010).Application of several micromolar ABA also establishes tolerance to rapid desiccation by exposing to an atmosphere of about à100MPa(about50%relative humidity)in a laminar?ow cabinet for24h (Khandelwal et al.2010).Osmostress also induces accumulation of endogenous
ABA in P.patens(Minami et al.2005).These facts also indicate a role for ABA in
induction of DT in P.patens.Furthermore,exogenous ABA induces accumulation
of boiling-soluble proteins in P.patens,a characteristic feature of hydrophilic LEA
proteins which speci?cally accumulate in developing seeds of angiosperms
(Knight et al.1995).ABA treatment of P.patens also induces accumulation of
soluble sugars such as sucrose(Oldenhof et al.2006;Nagao et al.2006)and a
trisaccharide theanderose(Nagao et al.2006).Oldenhof et al.(2006)reported that
exogenous ABA preserves membrane phase behavior in the dried state and that the
membranes do not undergo a gel to liquid membrane phase transition during
dry–rehydration process.It is likely that ABA functions to elicit cellular protection
mechanisms upon the rehydration phase to regain integrity of membranes.
The Arabidopsis abi3mutant lacks expression of a set of seed-speci?c genes
including LEA and desiccation tolerance of seeds(Nambara et al.1995;Marella et al.
74Y.Sakata et al. 2006),indicating that ABA-and ABI3-mediated gene expression is key for desiccation tolerance of seeds.The ABA-and ABI3-mediated regulation of LEA gene expression is evolutionarily conserved between P.patens and Arabidopsis(Marella et al.2006). Moreover,disruption of ABI3results in loss of ABA-induced DT of P.patens (Khandelwal et al.2010).These data collectively suggest that the mechanism that confers desiccation tolerance of plant cells was established in the last common ancestor of bryophytes and angiosperms.During evolution,land plants acquired a way to execute the induction of desiccation tolerance in an ABA-dependent manner,and the system independently has been used to provide desiccation tolerance of vegetative tissues of bryophytes and angiosperms seeds.
4.2Low Temperature Responses
Overwintering plants in the temperate and sub-frigid zones of earth in general can acclimate to low temperatures,and these plants develop freezing tolerance in response to pre-exposure to nonfreezing low temperature.It has been suggested that ABA plays a role in this cold acclimation process.Endogenous ABA levels increase during cold acclimation(Daie and Campbell1981;Lalk and Dorf?ing 1985;Smolen′ska-Sym et al.1995;Jouve et al.2000),and exogenous ABA treat-ment at ambient temperatures increases freezing tolerance(Chen and Gusta1983). Analysis of transcripts has indicated that ABA treatment increases expression of a number of stress-responsive genes that are also induced by cold(Zeevaart and Creelman1988).These results suggest that the endogenous ABA accumulated in response to low temperature exposure plays a role in enhanced freezing tolerance. Accordingly,analysis of cold-induced genes of Arabidopsis has revealed the frequent occurrence of ABRE in the promoters(Yamaguchi-Shinozaki and Shinozaki1994;Seki et al.2002).
Despite these facts,the role of ABA in cold acclimation has yet to be conclusively established(Gusta et al.2005).Studies on ABA-de?cient aba1and ABA-insensitive abi1-1of Arabidopsis have indicated that the contribution of ABA to cold acclimation might be limited.Both ABA-de?cient aba1and ABA-insensitive abi1-1mutants showed accumulation of cold responsive(COR)transcripts by low temperature treatment(Gilmour and Thomashow1991).In addition,cold-induced gene expression is mediated by CBF/DREB transcription factors that bind to the distinct cis-element C-repeat,also known as the dehydration-responsive element(DRE),independent of ABA(Baker et al.1994).These results suggest that endogenous ABA has little or no role in the induction of cold tolerance.However,the issue is not simple because the C-repeat/DRE can serve as a coupling element(CE)with ABRE;thus,the C-repeat/ DRE contributes to the ABA-induced gene expression when present with ABRE (Narusaka et al.2003).In addition,expression of genes for CBFs/DREBs is induced by ABA(Knight et al.2004).In contrast to molecular studies of cold responses in Arabidopsis,such studies on other plant species with various cold tolerances are limited.Recent global promoter analyses indicate that both ABRE and C-repeat/ DRE are conserved in cold-induced promoters of Arabidopsis and soybean.ABRE
ABA as a Universal Plant Hormone75 is also conserved in the cold-induced promoter of rice,but the C-repeat/DRE is not (Maruyama et al.2012).These results indicate that the mechanism for cold-induced gene expression might vary even among angiosperm species,though implicating the presence of evolutionarily conserved cis-promoter elements regulated by ABA.
Even nonvascular plants can undergo cold acclimation.Moss species growing in their natural habitat have a higher freezing tolerance in winter than in summer (Ru¨tten and Santarius1992).Laboratory experiments using P.patens have indicated that its protonemata and gametophore develop freezing tolerance in response to exposure to nonfreezing cold temperatures for several days(Minami et al.2005; Sun et al.2007).The increase in freezing tolerance accompanies an increase in various LEA-like transcripts,which are also inducible by ABA treatment.P.patens genes,which are remarkably induced by cold acclimation,contain putative ABREs but not C-repeats/DRE in the promoter,indicating a possible role of evolutionarily conserved cis-elements in cold-induced gene expression(Cuming et al.2007; Bhyan et al.2012).In contrast to these facts,analysis of endogenous ABA levels has suggested that obvious increases in ABA levels are not observed during up to one week of cold acclimation(Minami et al.2005).Thus,the contribution of endogenous ABA in cold acclimation of moss is not yet conclusive.
Our recent results using ABA-insensitive AR7lines of P.patens indicate that the ABA signaling process is necessary for cold responses in mosses.The AR7mutant that has been isolated by ultraviolet mutagenesis and the D2-1line that overexpresses the catalytic domain of Group A PP2C show an ABA-insensitive growth and cannot develop freezing and desiccation tolerance.Of interest,these lines did not acclimate to low temperature to enhance freezing tolerance,with reduced accumulation of stress-associated transcripts(Bhyan et al.2012).These results indicate that cold and ABA signal transduction share a common mechanism regulated by reversible phosphorylation.Identi?cation of the molecule under such regulation as well as transcription factors responsible for the cold-induced gene expression would clarify how ABA signaling molecules modify the cold-response pathway.
4.3Stomatal Closure
Stomata are widely distributed among land plants except liverworts,indicating that the epidermal apparatus evolved after the separation of liverworts and mosses from a common ancestor more than400million years ago(Fig.1).The role of ABA in seed plant stomatal closure is well documented(Schroeder et al.2001;Rock et al. 2010;Mori and Murata2011).Water-stressed seed plants increase accumulation of ABA,which effectively closes the stomatal aperture by reducing the turgor pressure of guard cells.This physiological event is considered as one of the most important functions of ABA to protect plants from water de?ciency.However,ABA signaling can be found in liverworts(Tougane et al.2010),suggesting that the role of ABA in stomatal closure is an evolutionarily acquired feature.It is still a matter of debate as to when land plants acquired the ABA-dependent closure system of the stomatal aperture in their evolutionary history;one group has reported that ABA
76Y.Sakata et al. involvement in regulating plant water loss and hydration through stomata is found only in seed plants,but another group has suggested that ABA in?uences the guard cell aperture of the basal vascular plants.Brodribb and McAdam(2011)examined the ABA response of the stomata of lycophytes and ferns by adding a high concentration of ABA to the transpiration stream.Even with the high concentration of foliar ABA(7,000ng gà1FW),stomata of fern(Pteridium esculentum)and lycophyte(Lycopodium deuterodensum)species lacked the closure response, although angiosperm(Helianthus annuus)and conifer(Callitris rhomboidea)spe-cies showed rapid stomatal closure at leaf ABA levels of1,500–2,000ng gà1 FW.Moreover,they showed a strong correlation between leaf water content and stomatal aperture in lycophytes and ferns.These authors suggested that stomatal closure of lycophytes and ferns is passively controlled and insensitive to ABA and that active metabolic control of stomatal closure evolved after the divergence of ferns about360million years ago.However,this hypothesis has been challenged (Ruszala et al.2011).They examined stomatal responses of the lycophyte Sellaginella uncinata to ABA as well as CO2in comparison with Arabidopsis. Exogenous ABA from0to25μM inhibited the light-induced opening of pre-closed stomata and promoted stomatal closure of pre-opened stomata dose dependently, although McAdam and Brodribb(2012)speci?cally examined this?nding and could not repeat it(see below).This ABA effect was blocked in the presence of EGTA and verapamil,which inhibit ABA-induced calcium transient in angiosperm guard cells.ABA also induced an increase of reactive oxygen species(ROS)in S.uncinata,the intracellular second messenger of the Ca2+transient in angiosperms guard cells.These authors further demonstrated that the S.uncinata gene encoding an SnRK2similar to Arabidopsis OST1could complement the Arabidopsis ost1 mutant phenotype.The stomata of S.uncinata also exhibited CO2-induced stomatal closure as observed in seed plants.These data suggested that evolutionarily conserved mechanisms operate stomatal closure between S.uncinata and angiosperms.According to this report,active stomatal control emerged in ancestral vascular plants about420million years ago.However,an evolutionarily conserved function of OST1-like SnRK2may not necessarily be related to the conserved mechanism of stomatal closure because this type of kinase can be found in the liverwort M.polymorpha,which lacks stomata(data not shown).
The con?ict between the reports may be attributed to the method or plant materials they employed;however,guard cells of ferns and lycophytes might possess a signaling pathway that can be activated by ABA.In fact,a set of genes that encode signaling components and biosynthesis enzymes for ABA in angiosperms is evolutionarily conserved among land plants including the basal land plant bryophytes that diverged from vascular plants420million years ago.It is intriguing that the known ABA transporter genes can be found only in seed plants (Table1).Acquisition of these genes in seed plants might contribute to the rapid reaction of the stomatal aperture in response to environmental water conditions.As mentioned?rst in this section,liverworts,which represent the oldest extant lineage of land plants,lack stomata but possess the ABA signaling pathway,indicating that ABA evolved as a phytohormone to regulate physiological aspects other than
信号转导 061M5007H 学期：2015-2016学年秋| 课程属性：| 任课教师：谢旗等 教学目的、要求 本课程为细胞生物学专业研究生的专业基础课，同时也可作为相关专业研究生的选修课。细胞信号转导是细胞生物学学科进展最快的研究领域之一，信号转导的概念已经开始深入到生命科学的各个领域。本课程内容涵盖动植物受体、G蛋白、环核苷酸第二信使、质膜磷脂代谢产物胞内信使、酶活性受体、蛋白质可逆磷酸化、泛素蛋白化及其对基因表达的调控、信号转导途径的多样性、网络化和专一性等方面的研究现状和进展。 预修课程 生物化学、分子生物学 教材 生命科学学院 主要内容 第一章绪论（3学时，教师：谢旗）细胞信号转导的研究对象和研究意义，细胞信号的主要种类，细胞化学信号分子与信号传递途径的特征。真核生物的蛋白激酶，蛋白磷酸酶，蛋白质可逆磷酸化对信号转导的调节方式，蛋白质可逆磷酸化与基因表达调控，蛋白质可逆磷酸化在细胞信号中的意义。蛋白质稳定性与信号转导。第二章植物免疫的表观遗传调控（3学时，教师：郭惠珊）表观遗传调控包含RNA干扰、DNA修饰、组蛋白翻译后修饰和染色质重塑等各种过程互相交叠，共同调控基因组表观修饰的动态平衡；除了影响生长和发育，表观遗传调控的另一重要功能是抗病免疫作用。本讲将着重介绍植物表观遗传途径及其抗病免疫信号的调控作用。第三章MicroRNA介导的信号（3学时，教师：郭惠珊）microRNA 广泛存在于生物体内，是生物体保守机制RNA沉默过程产生并具有序列特异性调控功能的一类非编码小分子RNA。本课程主要讲授植物microRNA的产生、加工、特性及其调控作用的基本生物学过程；以及植物miRNAs和其他小分子RNA参与植物生长素信号途径和其他植物生理性状的调控作用。第四章钙离子通道及信号转导（3学时，教师：陈宇航）钙离子是生命活动的必需元素，基本分布和内稳，代谢平衡和疾病；钙离子发挥重要生物学功能，简述历史发现，作为第二信使的化学基础，功能调控的基本模式，以钙结合蛋白为例子展开介绍钙离子发挥功能调控的分子结构基础等；介绍钙离子信号转导系统的组成，
细胞信号转导 李婧 2015212351 一、名词解释 内分泌 接触依赖性通讯 受体 G蛋白 第二信使 二、单项选择题 1、下列不属于信号分子的是 A.K+ B.cAMP C. cGMP D.Ca2+ 2、下列那个不是信号转导系统的主要特性 A.特异性 B.放大效应 C.整合作用 D.传递作用 3、（）是细胞表面受体中最大的多样性家族 A.G蛋白偶联受体 B.RTK C.Notch D.细胞因子 4、G蛋白偶联受体中（）是分子开关蛋白 A.G α B.Gβ C.GΘ D.Gγ 5、G蛋白偶联的光敏感受体的活化诱发（）的关闭 A.cAMP–PKA信号通路 B.Notch信号通路 C.JAK-STAT信号通路 D. cGMP门控阳离子通道 6、（）信号对细胞内糖原代谢起关键调控作用 -Ca2+ B.DAG-PKC C. cAMP–PKA D.RTK-Ras A.IP 3 7、（）的主要功能是引发贮存在内质网中的Ca2+转移到细胞质基质中，使 胞质中游离Ca2+浓度提高 B.PIP2 C.DAG D.PKC A. IP 3 8、（）主要功能是控制细胞生长、分化，而不是调控细胞中间代谢 A.RTK B. PKC C.PKB D.Wnt 9、Hedgehog信号通路控制 A.糖原代谢 B.细胞凋亡 C.细胞分化 D.氨基酸代谢 10、细胞通过配体依赖性的受体介导的（）减少细胞表面可利用受体数目。 A. 抑制性蛋白产生 B.内吞作用 C.敏感性下调 D.消化降解 三、多项选择题 1、细胞分泌化学信号可长距离或短距离发挥作用，其作用方式分为 A.内分泌 B.旁分泌 C.通过化学突出传递神经信号 D.外分泌 2、下列（）是糖脂 A.霍乱毒素受体 B.百日咳的毒素受体 C.甲状腺受体 D.胰岛素受体 3、下面关于受体酪氨酸激酶的说法正确的是 A.是一种生长因子类受体 B.受体蛋白只有一次跨膜 C.与配体结合后两个受体相互靠近，相互激活 D.具有SH2结构域
植物生长发育的各个阶段, 包括胚胎发生、种子萌发、营养生长、果实成熟、叶片衰老等都受到多种植物激素信号的控制。人们对植物激素的生物合成途径、生理作用已有大量阐述，在生产上的应用也已取得很大进展，但对其信号转导途径的认识并不是很全面。今天小编和大家聊一聊，9大类植物激素信号转导途径。 1.生长素 与生长素信号转导相关的三类蛋白组分是:生长素受体相关SCF复合体(SKP1, Cullin and F-box complex)、发挥御制功能的生长素蛋白(Aux/IAA)和生长素响应因子(ARF)。早期响应基因有Aux/IAA基因家族、GH1、GH3、GH2/4、SAUR基因家族、ACS、GST。生长素信号转导通路主要有4条: TIR1/AFBAux/IAA/TPL-ARFs途径、T MK1-IAA32/34-ARFs途径、TMK1/ABP1-ROP2/6-PINs或RICs 途径和SKP2AE2FC/DPB途径。 2.细胞分裂素
细胞分裂素信号转导途径是基于双元信号系统（TCS），通过磷酸基团在主要组分之间的连续传递而实现。双元信号系统主要包含3类蛋白成员及4次磷酸化事件: (ⅰ)位于内质网膜或细胞膜的组氨酸受体激酶(histidine kinases, HKs)感知细胞分裂素后发生组氨酸的自磷酸化；(ⅱ)将组氨酸残基的磷酸基团转移至自身接受区的天冬氨酸残基上；(ⅲ)受体天冬氨酸残基上的磷酸基团转移至细胞质的组氨酸磷酸化转移蛋白(His-containing phosphotransfer protein, HPs)的组氨酸残基上；(ⅳ)磷酸化的组氨酸转移蛋白进入细胞核并将磷酸基团转移至A类或B类响应调节因子(response regulators, ARR s)。在拟南芥中已知的细胞分裂素受体有AHK2、AHK3和AHK4 3个，AHP有6个(AHP1?6)，A类和B类ARR分別有10个和1 2个，它们是细胞分裂素信号转导通路的主要组成部分。
第十五章细胞信号转导 复习测试 （一）名词解释 1. 受体 2. 激素 3. 信号分子 4. G蛋白 5. 细胞因子 6. 自分泌信号传递 7. 蛋白激酶 8. 钙调蛋白 9. G蛋白偶联型受体 10. 向上调节 11. 细胞信号转导途径 12. 第二信使 （二）选择题 A型题： 1. 关于激素描述错误的是： A. 由内分泌腺/细胞合成并分泌 B. 经血液循环转运 C. 与相应的受体共价结合 D. 作用的强弱与其浓度相关 E. 可在靶细胞膜表面或细胞内发挥作用 2. 下列哪种激素属于多肽及蛋白质类： A. 糖皮质激素 B. 胰岛素 C. 肾上腺素 D. 前列腺素 E. 甲状腺激素 3. 生长因子的特点不包括： A. 是一类信号分子 B. 由特殊分化的内分泌腺所分泌 C. 作用于特定的靶细胞 D. 主要以旁分泌和自分泌方式发挥作用 E. 其化学本质为蛋白质或多肽 4. 根据经典的定义，细胞因子与激素的主要区别是： A. 是一类信号分子 B. 作用于特定的靶细胞 C. 由普通细胞合成并分泌 D. 可调节靶细胞的生长、分化 E. 以内分泌、旁分泌和自分泌方式发挥作用 5. 神经递质、激素、生长因子和细胞因子可通过下列哪一条共同途径传递信号：
A. 形成动作电位 B. 使离子通道开放 C. 与受体结合 D. 通过胞饮进入细胞 E. 自由进出细胞 6. 受体的化学本质是： A. 多糖 B. 长链不饱和脂肪酸 C. 生物碱 D. 蛋白质 E. 类固醇 7. 受体的特异性取决于： A. 活性中心的构象 B. 配体结合域的构象 C. 细胞膜的流动性 D. 信号转导功能域的构象 E. G蛋白的构象 8. 关于受体的作用特点，下列哪项是错误的： A. 特异性较高 B. 是可逆的 C. 其解离常数越大，产生的生物效应越大 D. 是可饱和的 E. 结合后受体可发生变构 9. 下列哪项与受体的性质不符： A. 各类激素有其特异性的受体 B. 各类生长因子有其特异性的受体 C. 神经递质有其特异性的受体 D. 受体的本质是蛋白质 E. 受体只存在于细胞膜上 10. 下列哪种受体是催化型受体： A. 胰岛素受体 B. 甲状腺激素受体 C. 糖皮质激素受体 受体 D. 肾上腺素能受体 E. 活性维生素D 3 11. 酪氨酸蛋白激酶的作用是： A. 使蛋白质结合上酪氨酸 B. 使含有酪氨酸的蛋白质激活 C. 使蛋白质中的酪氨酸激活 D. 使效应蛋白中的酪氨酸残基磷酸化 E. 使蛋白质中的酪氨酸分解 12. 下列哪种激素的受体属于胞内转录因子型： A. 肾上腺素 B. 甲状腺激素 C. 胰岛素 D. 促甲状腺素 E. 胰高血糖素
第十五章细胞信号转导 复习测试（一）名词解释 1. 受体 2. 激素 3. 信号分子 4. G蛋白 5. 细胞因子 6. 自分泌信号传递 7. 蛋白激酶 8. 钙调蛋白 9. G蛋白偶联型受体 10. 向上调节 11. 细胞信号转导途径 12. 第二信使 （二）选择题 A型题： 1. 关于激素描述错误的是： A. 由内分泌腺/细胞合成并分泌 B. 经血液循环转运 C. 与相应的受体共价结合 D. 作用的强弱与其浓度相关 E. 可在靶细胞膜表面或细胞内发挥作用 2. 下列哪种激素属于多肽及蛋白质类： A. 糖皮质激素 B. 胰岛素 C. 肾上腺素 D. 前列腺素 E. 甲状腺激素 3. 生长因子的特点不包括： A. 是一类信号分子 B. 由特殊分化的内分泌腺所分泌 C. 作用于特定的靶细胞 D. 主要以旁分泌和自分泌方式发挥作用
E. 其化学本质为蛋白质或多肽 4. 根据经典的定义，细胞因子与激素的主要区别是： A. 是一类信号分子 B. 作用于特定的靶细胞 C. 由普通细胞合成并分泌 D. 可调节靶细胞的生长、分化 E. 以内分泌、旁分泌和自分泌方式发挥作用 5. 神经递质、激素、生长因子和细胞因子可通过下列哪一条共同途径传递信号： A. 形成动作电位 B. 使离子通道开放 C. 与受体结合 D. 通过胞饮进入细胞 E. 自由进出细胞 6. 受体的化学本质是： A. 多糖 B. 长链不饱和脂肪酸 C. 生物碱 D. 蛋白质 E. 类固醇 7. 受体的特异性取决于： A. 活性中心的构象 B. 配体结合域的构象 C. 细胞膜的流动性 D. 信号转导功能域的构象 E. G蛋白的构象 8. 关于受体的作用特点，下列哪项是错误的： A. 特异性较高 B. 是可逆的 C. 其解离常数越大，产生的生物效应越大 D. 是可饱和的 E. 结合后受体可发生变构 9. 下列哪项与受体的性质不符： A. 各类激素有其特异性的受体 B. 各类生长因子有其特异性的受体 C. 神经递质有其特异性的受体 D. 受体的本质是蛋白质
细胞信号转导途径研究方法 一、蛋白质表达水平和细胞内定位研究 1、信号蛋白分子表达水平及分子量检测: Western blot analysis. 蛋白质印迹法是将蛋白质混合样品经SDS-PAGE后,分离为不同条带，其中含有能与特异性抗体(或McAb)相应的待检测的蛋白质(抗原蛋白)，将PAGE胶上的蛋白条带转移到NC 膜上此过程称为blotting，以利于随后的检测能够的进行，随后,将NC膜与抗血清一起孵育，使第一抗体与待检的抗原决定簇结合(特异大蛋白条带)，再与酶标的第二抗体反应,即检测样品的待测抗原并可对其定量。 基本流程： 检测示意图： 2、免疫荧光技术Immunofluorescence （IF） 免疫荧光技术是根据抗原抗体反应的原理，先将已知的抗原或抗体标记上荧光素制成荧光标记物，再用这种荧光抗体（或抗原）作为分子探针检查细胞或组织内的相应抗原（或抗体）。在细胞或组织中形成的抗原抗体复合物上含有荧光素，利用荧光显微镜观察标本，
荧光素受激发光的照射而发出明亮的荧光（黄绿色或桔红色），可以看见荧光所在的细胞或组织，从而确定抗原或抗体的性质、定位，以及利用定量技术测定含量。 采用流式细胞免疫荧光技术（FCM）可从单细胞水平检测不同细胞亚群中的蛋白质分子，用两种不同的荧光素分别标记抗不同蛋白质分子的抗体，可在同一细胞内同时检测两种不同的分子（Double IF），也可用多参数流式细胞术对胞内多种分子进行检测。 二、蛋白质与蛋白质相互作用的研究技术 1、免疫共沉淀（Co- Immunoprecipitation, Co-IP） Co-IP是利用抗原蛋白质和抗体的特异性结合以及细菌蛋白质的“protein A”能特异性地结合到免疫球蛋白的FC片段的现象而开发出来的方法。目前多用精制的protein A预先结合固化在agarose的beads上，使之与含有抗原的溶液及抗体反应后，beads上的prorein A 就能吸附抗原抗体达到沉淀抗原的目的。 当细胞在非变性条件下被裂解时，完整细胞内存在的许多蛋白质－蛋白质间的相互作用被保留了下来。如果用蛋白质X的抗体免疫沉淀X，那么与X在体内结合的蛋白质Y也能沉淀下来。进一步进行Western Blot和质谱分析。这种方法常用于测定两种目标蛋白质是否在体内结合，也可用于确定一种特定蛋白质的新的作用搭档。缺点：可能检测不到低亲和力和瞬间的蛋白质－蛋白质相互作用。 2、G ST pull-down assay GST pull-down assay是将谷胱甘肽巯基转移酶（GST）融合蛋白（标记蛋白或者饵蛋白，GST, His6, Flag, biotin …）作为探针，与溶液中的特异性搭档蛋白(test protein或者prey被扑获蛋白)结合，然后根据谷胱甘肽琼脂糖球珠能够沉淀GST融合蛋白的能力来确定相互作用的蛋白。一般在发现抗体干扰蛋白质－蛋白质之间的相互作用时，可以启用GST沉降技术。该方法只是用于确定体外的相互作用。 示意图：
第十一章细胞的信号转导 一、名词解释 1、细胞通讯 2、受体 3、第一信使 4、第二信使 5、G 蛋白 6、蛋白激酶A 二、填空题 1、细胞膜表面受体主要有三类即、、和。 2、在细胞的信号转导中，第二信使主要有、、、和。 3、硝酸甘油之所以能治疗心绞痛是因为它在体内能转化为，引起血管，从而减轻的负荷和的需氧量。 三、选择题 1、能与胞外信号特异识别和结合，介导胞内信使生成，引起细胞产生效应的是( )。 A、载体蛋白 B、通道蛋白 C、受体 D、配体 2、下列不属于第二信使的是（）。 A、cAMP B、cGMP C、DG D、CO 3、下列关于信号分子的描述中，不正确的一项是（）。 A、本身不参与催化反应 B、本身不具有酶的活性 C、能够传递信息 D、可作为酶作用的底物 4、生长因子是细胞内的（）。 A、结构物质 B、能源物质 C、信息分子 D、酶 5、肾上腺素可诱导一些酶将储藏在肝细胞和肌细胞中的糖原水解，第一个被激活的酶是（）。 A、蛋白激酶A B、糖原合成酶 C、糖原磷酸化酶 D、腺苷酸环化酶 6、（）不是细胞表面受体。 A、离子通道 B、酶连受体 C、G蛋白偶联受体 D、核受体 7、动物细胞中cAMP的主要生物学功能是活化（）。 A、蛋白激酶C B、蛋白激酶A C、蛋白激酶K D、Ca2+激酶 8、在G蛋白中，α亚基的活性状态是（）。 A、与GTP结合，与βγ分离 B、与GTP结合，与βγ聚合 C、与GDP结合，与βγ分离 D、与GDP结合，与βγ聚合
9、下面关于受体酪氨酸激酶的说法哪一个是错误的 A、是一种生长因子类受体 B、受体蛋白只有一次跨膜 C、与配体结合后两个受体相互靠近，相互激活 D、具有SH2结构域 10、在与配体结合后直接行使酶功能的受体是 A、生长因子受体 B、配体闸门离子通道 C、G蛋白偶联受体 D、细胞核受体 11、硝酸甘油治疗心脏病的原理在于 A、激活腺苷酸环化酶，生成cAMP B、激活细胞膜上的GC，生成cGMP C、分解生成NO，生成cGMP D、激活PLC，生成DAG 12、霍乱杆菌引起急性腹泻是由于 A、G蛋白持续激活 B、G蛋白不能被激活 C、受体封闭 D、蛋白激酶PKC功能异常 13下面由cAMP激活的酶是 A、PTK B、PKA C、PKC D、PKG 14下列物质是第二信使的是 A、G蛋白 B、NO C、GTP D、PKC 15下面关于钙调蛋白（CaM）的说法错误的是 A、是Ca2+信号系统中起重要作用 B、必须与Ca2+结合才能发挥作用 C、能使蛋白磷酸化 D、CaM激酶是它的靶酶之一16间接激活或抑制细胞膜表面结合的酶或离子通道的受体是 A、生长因子受体 B、配体闸门离子通道 C、G蛋白偶联受体 D、细胞核受体 17重症肌无力是由于 A、G蛋白功能下降
生物膜与细胞信号转导 名词解释： 1.脂筏：胆固醇分子不可能在脂双层里均匀分布，而是与鞘脂一起集中在膜的 特定区域，胆固醇-鞘脂漂浮在液态磷酸甘油脂“海洋”上的“筏”一样称为脂筏。 2.转运蛋白： 3.P-型ATPase：是阳离子转运蛋白，在转运过程中需要ATP可逆磷酸化的过 程，磷酸化使得转运蛋白的构象发生变化，同时，转运阳离子做跨膜运输4.次级主动运输：第一种溶质(S1)通过初级主动运输产生浓度梯度后，接着， 第一种溶质顺着浓度梯度提供能量，驱动第二种溶质(S2)逆浓度梯度运输 5.G蛋白分子开关：GTP酶(GTPase)是一个分子开关，开关是通过结合和水解 GTP进行控制。 6.激酶锚定蛋白：AKAPs 是支架蛋白，位于脂筏的胞质侧，将信号通路中执 行功能的蛋白聚集在一起，便于反应进行 7.信号蛋白 8.MAP激酶级联反应：酵母中的mating pheromones，果蝇中复眼的光受体的 分化，开花植物中对病源的防御反应。 简答题： 1.溶质分子跨膜运动，有哪几种机制？ 2.以细菌KcsA钾离子通道为例，说明电压门控的钾离子通道结构与运输的关系答：细菌KcsA通道是由四个亚基组成，其中两个亚基由两个跨膜的螺旋(M1和M2) 和通道胞外的孔区域(P)组成。每个亚基的M2螺旋线与另一个亚基的M2相互交叉形成一个“螺旋束”，封闭了面向质膜的孔，则K+不能通过；M2 螺旋线可以在具有甘氨酸残基的位点弯曲，将通道门打开。K+运输。 P区域是由一个长约1/3通道宽度的短的螺旋和一个能形成“衬里的”狭窄的选择性过滤器的无螺旋的环，允许K+通过。选择性过滤器的衬里含有高度保守的五肽骨架，产生5个连续排列的氧原子环。每个环由四个氧原子组成，直径是3nm，而失水K+直径是2.7nm。当通道门打开，K+进入通道时，电负性的氧原子替代了与K+结合的水分子，与K+稳定的相互作用使K+运输。尽管选择性过滤器具有4个K+结合位点，但实际上只能同时结合2个K+。 3.乙酰胆碱受体门控通道结构及离子运输机制 结构：乙酰胆碱是由运动神经元释放到肌细胞质膜，与乙酰胆碱受体结合，它可以改变受体的构象，引起离子通道打开。乙酰胆碱受体允许Na+、Ca2+和K+通过。由5个亚基组成：γ，β和δ各1个，2个α亚基，每个α亚基带有1个乙酰胆碱结合位点。每个亚基含有4个跨膜双螺旋，5个亚基围成1个中心孔，孔直径约20 ?，突出在胞质和细胞表面。 机制：2个乙酰胆碱结合到2个α亚基上，引起构象发生变化，使疏水侧链远离通道的中心，打开离子通道，让离子通过。组成5个亚基的M2螺旋所含有的5个Leu 侧链突出在通道，限制了通道的直径。当两个乙酰胆碱受体位点被占据，构想发生变化，随着M2螺旋的轻微扭曲，5个Leu残基旋转，远离通道中心，由较小的极性氨基酸代替，通过道门打开，允许Ca2+，Na+、K+通过。
细胞受体类型、特点 及重要的细胞信号转导途径 学院：动物科学技术学院 专业：动物遗传育种与繁殖 姓名：李波
学号：2015050509
目录 1、细胞受体类型及特点 (4) 1.1离子通道型受体 (4) 1.2 G蛋白耦联型受体 (4) 1.3 酶耦联型受体 (5) 2、重要的细胞信号转导途径 (5) 2.1细胞内受体介导的信号传递 (5) 2.2 G蛋白偶联受体介导的信号转导 (6) 2.2.1激活离子通道的G蛋白偶联受体所介导的信号通路 (7) 2.2.2激活或抑制腺苷酸环化酶的G蛋白偶联受体 (7) 2.2.3 激活磷脂酶C、以lP3和DAG作为双信使 G蛋白偶联受体介导的信号通 路 (8) 2.2 酶联受体介导的信号转导 (9) 2.2.1 受体酪氨酸激酶及RTK-Ras蛋白信号通路 (10) 2.2.2 P13K-PKB(Akt)信号通路 (10) 2.2.3 TGF-p—Smad信号通 (11) 2.2.4 JAK—STAT信号通路 (12)
1、细胞受体类型及特点 受体(receptor)是一种能够识别和选择性结合某种配体(信号分子)的大分子物质，多为糖蛋白，一般至少包括两个功能区域，与配体结合的区域和产生效应的区域，当受体与配体结合后，构象改变而产生活性，启动一系列过程，最终表现为生物学效应。受体与配体问的作用具有3个主要特征：①特异性；②饱和性；③高度的亲和力。 根据靶细胞上受体存在的部位，可将受体分为细胞内受体(intracellular receptor)和细胞表面受体(cell surface receptor)。细胞内受体介导亲脂性信号分子的信息传递，如胞内的甾体类激素受体。细胞表面受体介导亲水性信号分子的信息传递，膜表面受体主要有三类：①离子通道型受体(ion—channel—linked receptor)；②G蛋白耦联型受体(G—protein —linked receptor)；③酶耦联的受体(enzyme—linked recep—tor)。第一类存在于可兴奋细胞。后两类存在于大多数细胞，在信号转导的早期表现为激酶级联事件，即为一系列蛋白质的逐级磷酸化，借此使信号逐级传送和放大。 1.1离子通道型受体 离子通道型受体是一类自身为离子通道的受体，即配体门通道(1igand—gated channel)，主要存在于神经、肌肉等可兴奋细胞，其信号分子为神经递质。神经递质通过与受体的结合而改变通道蛋白的构象，导致离子通道的开启或关闭，改变质膜的离子通透性，在瞬间将胞外化学信号转换为电信号，继而改变突触后细胞的兴奋性。如：乙酰胆碱受体以三种构象存在，两分子乙酰胆碱的结合可以使之处于通道开放构象，但该受体处于通道开放构象状态的时限仍十分短暂，在几十毫微秒内又回到关闭状态。然后乙酰胆碱与之解离，受体则恢复到初始状态，做好重新接受配体的准备。离子通道型受体分为阳离子通道，如乙酰胆碱、谷氨酸和五羟色胺的受体，和阴离子通道。 1.2 G蛋白耦联型受体 三聚体GTP结合调节蛋白(trimeric GTP—binding regulatory protein)简称G蛋白，位于质膜胞质侧，由a、p、-／三个亚基组成，a和7亚基通过共价结合的脂肪酸链尾结合在膜上，G蛋白在信号转导过程中起着分子开关的作用，当a亚基与GDP结合时处于关闭状态，与GTP结合时处于开启状态，“亚基具有GTP酶活性，能催化所结合的ATP 水解，恢复无活性的三聚体状态，其GTP酶的活性能被RGS(regulator of G protein signaling)增强。RGS也属于GAP(GTPase activating protein)。 G蛋白耦联型受体为7次跨膜蛋白(图10—6)，受体胞外结构域识别胞外信号分子并与之结合，胞内结构域与G蛋白耦联。通过与G蛋白耦联，调节相关酶活性，在细胞内
表皮生长因子两条典型的信号转导途径 (1)表皮生长因子受体介导的信号转导途径 表皮生长因子与其受体－表皮生长因子受体结合后可引发一系列细胞内变化，最终使细胞发生分化或增殖。表皮生长因子受体是一种受体酪氨酸蛋白激酶，而受体酪氨酸蛋白激酶→Ras→MAPK级联途径是表皮生长因子刺激信号传递到细胞核内的最主要途径。它由以下成员组成：表皮生长因子受体→含有SH2结构域的接头蛋白(如Grb2)→鸟嘌呤核苷酸释放 因子(如SOS)→Ras蛋白→MAPKKK(如Raf1)→MAPKK→MAPK→转录因子等(图21－24)。 表皮生长因子与受体结合后，可以使受体发生二聚体化，从而改变了受体的构象，使其中的蛋白酪氨酸激酶活性增强，受体自身的酪氨酸残基发生磷酸化，磷酸化的受体便形成了与含SH2结构域的蛋白分子Grb2结合的位点，导致Grb2与受体的结合。Grb2中有两个SH3结构域，该部位与一种称为SOS的鸟苷酸交换因子结合，使之活性改变，SOS则进一步活化Ras，激活的Ras作用于MAPK激活系统，导致ERK的激活。最后ERK转移到 细胞核内，导致某些转录因子的活性改变从而改变基因的表达状态及细胞的增殖与分化过程。 (2)γ－干扰素受体介导的信号转导 γ－干扰素是由活化T细胞产生的，它具有促进抗原提呈和特异性免疫识别的作用，并可促进B细胞分泌抗体。γ－干扰素与受体结合以后，也可以导致受体二聚体化，二聚体化的受体可以激活JAK－STAT系统，后者将干扰素刺激信号传入核内。JAK(Janus Kinase)为一种存在于胞浆中的蛋白酪氨酸激酶，它活化后可使干扰素受体磷酸化。STAT(Signal Transducerand Activator of Transcription)可以通过其SH2结构域识别磷酸化的受体并与之结合。然后STAT分子亦发生酪氨酸的磷酸化，酪氨酸磷酸化的STAT进入胞核形成有活性的转录因子，影响基因的表达 表皮生长因子（epidermal growth factor，EGF）受体是研究得比较清楚的酪氨酸激酶受体，存在于特殊的靶细胞的质膜上，调节不同的功能，包括细胞的生长、增殖和分化，并且与肿瘤的发生有关。EGF受体（EGF receptor）结构是一种糖蛋白，由三个部分组成：①细胞外结构域有621个氨基酸残基，富含半胱氨酸，并形成多对二硫键。其上结合有糖基，是EGF结合的位点；②跨膜区由23个氨基酸残基组成；③细胞质结构域，由542个氨基酸残基组成，含有无活性的酪氨酸激酶和几个酪氨酸磷酸化的位点。当EGF同受体细胞外结构域结合位点结合后，受体被激活，导致两个EGF受体单体形成二聚体，激活细胞质部分的酪氨酸激酶，使酪氨酸自我磷酸化。EGF受体上有五个主要的磷酸化的酪氨酸位点，可以同几种不同的蛋白质结合，分别引起细胞内不同的信号应答。在多数情况下，EGF受体被磷酸化的酪氨酸位点同靶蛋白（酶）的SH2结构域相互作用，将靶蛋白（酶）激活，引起细胞应答。如PIP2激酶通过SH2与EGF受体磷酸化的酪氨酸位点相互作用被激活，激活的PIP2激酶使一种膜脂-PIP2磷酸化。另外，磷酸化的酪氨酸位点也可以同具有SH2结构域的磷脂酶Cγ相互作用，并将磷脂酶Cγ激活，激活的磷脂酶Cγ可将质膜中PIP2水解成IP3和DAG，引起与磷脂肌醇-G蛋白偶联系统类似的信号转导。
第三节主要的信号转导途径 一、膜受体介导的信号传导 （一）cAMP-蛋白激酶A途径 述：该途径以靶细胞内cAMP浓度改变和激活蛋白激酶A（PKA）为主要特征，是激素调节物质代谢的主要途径。 1．cAMP的合成与分解 ⑴引起cAMP水平增高的胞外信号分子：胰高血糖素、肾上腺素、 促肾上腺皮质激素、促甲状腺素、甲状旁腺素和加压素等。 α-GDP-βγ（Gs蛋白）激素+受体→激素-受体→↓ α-GTP + βγ ↓ AC激活 ↓ ATP →cAMP 述：当信号分子（胰高血糖素、肾上腺素和促肾上腺皮质激素）与靶细胞质膜上的特异性受体结合，形成激素一受体复合物 而激活受体。活化的受体可催化Gs的GDP与GTP交换，导 致Gs的α亚基与βγ解离，蛋白释放出αs-GTP。αs-GTP能激 活腺苷酸环化酶，催化ATP转化成cAMP，使细胞内cAMP 浓度增高。过去认为G蛋白中只有α亚基发挥作用，现知βγ 复合体也可独立地作用于相应的效应物，与α亚基拮抗。 腺苷酸环化酶分布广泛，除成熟红细胞外，几乎存在于所有组织的细胞质膜上。cAMP经磷酸二酯酶（PDE）降解成 5'－AMP而失活。cAMP是分布广泛而重要的第二信使。
⑵AC活性的抑制与cAMP浓度降低 ◇Gα-GTP结合AC并使之激活后，同时激活自身的GTP酶活性，Gα-GTP→Gα-GDP，Gs、AC均失活。从而在细胞对cAMP浓度升高作出应答后AC活性迅速逆转。 ⑶少数激素，如生长激素抑制素、胰岛素和抗血管紧张素II 等，它们活化受体后可催化抑制性G蛋白解离，导致细胞内AC活性下降，从而降低细胞内cAMP水平。 ⑷正常细胞内cAMP的平均浓度为10－6mol/L。cAMP在细 胞中的浓度除与腺苷酸环化酶活性有关外，还与磷酸二酯酶的活性有关。举例如下： ①一些激素如胰岛素，能激活磷酸二酯酶，加速cAMP降解； ②某些药物如茶碱，则抑制磷酸二酯酶，促使细胞内cAMP 浓度升高。 2．cAMP的作用机制――cAMP激活PKA（幻灯64） ⑴cAMP对细胞的调节作用是通过激活cAMP依赖性蛋白激酶 或称蛋白激酶A （PKA）系统来实现的。 ⑵PKA的结构 2C（催化亚基）：蛋白丝/苏氨酸磷酸化酶活性四聚体蛋白 变构酶 2R（调节亚基）：各有2个cAMP结合位点述：催化亚基有催化底物蛋白质某些特定丝／苏氨酸残基磷酸化的功能。调节亚基与催化亚基相结合时，PKA呈无活性状态。当4分子cAMP与2个调节亚基结合后，调节亚基脱落，游离的催化亚基具有蛋白激酶活性。PKA的激活过程需要Mg2+。
第八章细胞信号转导 名词解释 1、蛋白激酶protein kinase 将磷酸基团转移到其他蛋白质上的酶，通常对其他蛋白质的活性具有调节作用。 2、蛋白激酶C protein kinase C 一类多功能的丝氨酸/苏氨酸蛋白激酶家族，可磷酸化多种不同的蛋白质底物。 3、第二信使second messenger 第一信使分子（激素或其他配体）与细胞表面受体结合后，在细胞内产生或释放到细胞内的小分子物质，如cAMP，IP3，钙离子等，有助于信号向胞内进行传递。 4、分子开关molecular switch 细胞信号转导过程中，通过结合GTP与水解GTP，或者通过蛋白质磷酸化与去磷酸化而开启或关闭蛋白质的活性。 5、磷脂酶C phospholipid C 催化PIP2分解产生1,4,5-肌醇三磷酸（IP3）和二酰甘油(DAG)两个第二信使分子。 6、门控通道gated channel 一种离子通道，通过构象改变使溶液中的离子通过或阻止通过。依据引发构象改变的机制的不同，门控通道包括电位门通道和配体门通道两类。 7、神经递质neurotransmitter 突触前端释放的一种化学物质，与突触后靶细胞结合，并改变靶细胞的膜电位。 8、神经生长因子nerves growth factor，NGF 神经元存活所必需的细胞因子 9、受体receptor 任何能与特定信号分子结合的膜蛋白分子，通常导致细胞摄取反应或细胞信号转导。10、受体介导的胞吞作用receptor mediated endocytosis 通过网格蛋白有被小泡从胞外基质摄取特定大分子的途径。被转运的大分子物质与细胞表面互补性的受体结合，形成受体-配体复合物并引发细胞质膜局部内化作用，然后小窝脱离质膜形成有被小泡而将物质吞入细胞内。 11、受体酪氨酸激酶receptor tyrosine kinase，RTK 能将自身或胞质中底物上的酪氨酸残基磷酸化的细胞表面受体。主要参与细胞生长和分化的调控。 12、调节型分泌regulated secretion 细胞中已合成的分泌物质先储存在细胞质周边的分泌泡中，在受到适宜的信号刺激后，才与质膜融合将内容物分泌到细胞表面。 13、细胞通讯cell communication 信号细胞发出的信息传递到靶细胞并与受体相互作用，引起靶细胞产生特异性生物学效应的过程。 14、细胞信号传递cell signaling 通过信号分子与受体的相互作用，将外界信号经细胞质膜传递到细胞内部，通常传递至细胞核，并引发特异性生物学效应的过程。 15、信号转导signal transduction 细胞将外部信号转变为自身应答反应的过程。 16、组成型分泌constitutivesecretion
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PPAR-γ作用及其相关信号转导途径 作者：陈永熙， 王伟铭， 周同， 陈楠， Yong-Xi Chen， Wei-Ming Wang， Tong Zhou， Nan Chen 作者单位：上海交通大学医学院附属瑞金医院肾内科,上海,200032 刊名： 细胞生物学杂志 英文刊名：CHINESE JOURNAL OF CELL BIOLOGY 年，卷(期)：2006,28(3) 被引用次数：86次 参考文献(43条) 1.Rocchi S查看详情 1999 2.Sundvold H查看详情 2001 3.Rosen ED查看详情 2002 4.Fajas L查看详情 1998 5.Tautenhahn A查看详情 2003 6.Werman A查看详情 1997 7.Hah J查看详情 2000 8.Hsi LC查看详情 2001 9.Nolte RT查看详情 1998 10.Zingarelli B查看详情 2005 11.Li M查看详情 2000 12.Li Q查看详情 2002(02) 13.Bohrer H查看详情 1997 14.Ghosh S查看详情 1998 15.Straus DS查看详情 2000 16.Chung SW查看详情 2000 17.Sanchez-Hidalgo M查看详情 2005 18.Desvergne B查看详情 1999 19.Guan Y查看详情 2001 20.Tontonoz P查看详情 1998 21.Bruemmer D查看详情 2003 22.Delerive P查看详情 1999 23.Marx N查看详情 1999 24.Verrier E查看详情 2004 25.Fan WH查看详情 2000 26.Fu M查看详情 2001 27.Bendixen AC查看详情 2001 28.Goetze S查看详情 2002 29.Anandharajan R查看详情 2005 30.Hegyi K查看详情 2004
第十九章细胞信息转导的分子机制一、A型选择题 1．不作用于质膜受体的信息物质是 A．乙酰胆碱 D．甲状腺素 2．能激活PKG的是 A．cAMP A．脂类质 4．下列哪种物质能使蛋白质的酪氨酸残基发生磷酸化A．PKA B．PKC C．生长激素受体 D．类固醇激素受体 A．1个 A．1个 E．糖皮质激素受体 C．3个 C．3个 D．4个 D．4个 E．5个 E．5个 E．失活 B．cGMP B．糖类 C．Ca2+ D．DAG D．多肽 E．GTP E．蛋白 3．细胞膜受体的本质是 C．核酸 B．谷氨酸 E．神经酰胺 C．表皮生长因子 5．PKA中的每个调节亚基可结合cAMP的分子数为B．2个 B．2个
6．PKA所含的亚基数为 7．蛋白激酶的作用是使蛋白质或酶 A．磷酸化 B．脱磷酸化 C．水解 8．通过膜受体起调节作用的激素是 A．性激素 B．糖皮质激素 C．甲状腺素 9．下列关于GTP结合蛋白(G蛋白)的叙述,错误的是 （2007年全国硕士研究生入学考试西医综合科目试题） A．膜受体通过G蛋白与腺苷酸环化酶偶联 B．可催化GTP水解为GDP C．霍乱毒素可使其失活 D．有三种亚基α、β、γ D．激活 D．肾上腺素 E．活性维生素 E．G蛋白具有内源GTP酶活性 10．下列哪种酶激活后会直接引起cAMP浓度降低 （2006年全国硕士研究生入学考试西医综合科目试题） A．蛋白激酶A B．蛋白激酶C D．磷脂酶C E．蛋白激酶G 11．cAMP能别构激活下列哪种酶（2005年全国硕士研究生入学考试西医综合科目试题） A．磷脂酶A B．蛋白激酶A D．蛋白激酶G E．酪氨酸蛋白激酶 12．细胞膜内外正常Na+和K+浓度差的形成和维持是由于 （2004年全国硕士研究生入学考试西医综合科目试题） A．膜安静时K+通透性大 C．Na+易化扩散的结果 C．磷酸二酯酶 C．蛋白激酶C B．膜兴奋时Na+通透性增加 D．膜上Na+泵的作用 E．膜上Ca2+泵的作用