The endo-β-mannanase (MAN) family is represented in the Arabidopsis genome by eight members, all with canonical signal peptides and only half of them being expressed in germinating seeds. The transcripts of these genes were localized in the radicle and micropylar endosperm (ME) before radicle protrusion and this expression disappears as soon as the endosperm is broken by the emerging radicle tip. However, only three of these MAN genes, AtMAN5, AtMAN7 and especially AtMAN6 influence the germination time (t50) as assessed by the analysis of the corresponding knock-out lines. The data suggest a possible interaction between embryo and ME regarding the role of MAN during the Arabidopsis germination process

Angel J. Matilla

Arana MV

Cristina Barrero-Sicilia

Gong XM

Iglesias-Fernández R

Linkies A

Liu PP

María del Carmen Rodríguez-Gacio

Matilla AJ

Müller K

Nonogaki H

Pilar Carbonero

Raquel Iglesias-Fernández

Rodríguez-Gacio MC

Schröder R

Sliwinska E

Vicente-Carbajosa JV

Yuan JS

PubMed

The endo-β-mannanase (MAN) family is represented in the Arabidopsis genome by eight members, all with canonical signal peptides and only half of them being expressed in germinating seeds. The transcripts of these genes were localized in the radicle and micropylar endosperm (ME) before radicle protrusion and this expression disappears as soon as the endosperm is broken by the emerging radicle tip. However, only three of these MAN genes, AtMAN5, AtMAN7 and especially AtMAN6 influence the germination time (t50) as assessed by the analysis of the corresponding knock-out lines. The data suggest a possible interaction between embryo and ME regarding the role of MAN during the Arabidopsis germination process.



Iglesias-Fernández R, Rodríguez-Gacio MC, Barrero-Sicilia C, Carbonero P, Matilla A.J. Three endo-β-mannanase genes expressed in the micropylar endosperm and in the radicle influence germination of Arabidopsis thaliana seed

Iglesias Fernandez, Raquel

Rodriguez Gacio, Maria del Carmen

Barrero Sicilia, Cristina

Carbonero Zalduegui, Pilar

Matilla, Angel J.

Archivo Digital UPM

Molecular analysis of endo-beta-mannanase genes upon seed imbibition suggest a cross-talk between radicle and micropylar endosperm during  germination of Arabidopsis thaliana

Crossref

English

Molecular analysis of endo-β-mannanase genes upon seed imbibition suggest a cross-talk between radicle and micropylar endosperm during germination of
                    Arabidopsis thaliana

Molecular analysis of endo-β-mannanase genes upon seed imbibition suggest a cross-talk between radicle and micropylar endosperm during germination of Arabidopsis thaliana.

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©2011 Landes Bioscience.
Do not distribute.
www.landesbioscience.com Plant Signaling & Behavior 1
article addendum article addendum
Plant Signaling & Behavior 6:1, 1-3;  January 2011; © 2011 landes Bioscience
Addendum to: Iglesias-Fernández R, Rodríguez-
Gacio MC, Barrero-Sicilia C, Carbonero P, Matilla 
AJ. Three endo-β-mannanase genes expressed 
in the micropylar endosperm and in the radicle 
influence germination of Arabidopsis thaliana 
seeds. Planta 2010; In press; PMID: 20878180; DOI: 
10.1007/s00425-010-1257-z.
Key words: arabidopsis, endo-β-
mannanases, germination, in situ hybrid-
ization, micropylar endosperm, T-DNA 
insertion mutants
Submitted: 11/05/10
Accepted: 11/05/10
Previously published online: 
www.landesbioscience.com/journals/psb/
article/14126
DOI: 10.4161/psb/14126
*Correspondence to: Angel J. Matilla; 
Email: angeljesus.matilla@usc.es
The endo-β-mannanase (MAN) fam-ily is represented in the Arabidopsis 
genome by eight members, all with 
canonical signal peptides and only half 
of them being expressed in germinating 
seeds. The transcripts of these genes were 
localized in the radicle and micropylar 
endosperm (ME) before radicle protru-
sion and this expression disappears as 
soon as the endosperm is broken by the 
emerging radicle tip. However, only three 
of these MAN genes, AtMAN5, AtMAN7 
and especially AtMAN6 influence the 
germination time (t
50
) as assessed by the 
analysis of the corresponding knock-out 
lines. The data suggest a possible interac-
tion between embryo and ME regarding 
the role of MAN during the Arabidopsis 
germination process.
The Endosperm-limited Seed 
Germination
The seed phase is the most impor-
tant stage of the plant life cycle, ensur-
ing species survival. The seed is a viable 
and autonomous organism which will 
germinate when internal and environ-
ment conditions are suitable.1 In endo-
spermic seeds, the diploid embryo is 
surrounded by two covering layers: the 
triploid endosperm (nutritive tissue, liv-
ing cells) and the diploid testa (the seed 
coat, maternal tissue, dead cells). The 
endosperm of Arabidopsis is constituted 
by a single thin layer and originated by a 
Molecular analysis of endo-β-mannanase genes upon seed imbibition 
suggest a cross-talk between radicle and micropylar endosperm during 
germination of Arabidopsis thaliana
Raquel Iglesias-Fernández,2,† María del Carmen Rodríguez-Gacio,2 Cristina Barrero-Sicilia,1 Pilar Carbonero1 and Angel J. 
Matilla2,*
1Centro de Biotecnología y Genómica de Plantas (UPM-INIA); Universidad Politécnica de Madrid; Escuela Técnica Superior de Ingenieros Agrónomos; Campus 
de Montegancedo; Madrid; 2Departamento de Fisiología Vegetal; Facultad de Farmacia; Universidad de Santiago de Compostela; Santiago de Compostela, Spain
†Present address: Centro de Biotecnología y Genómica de Plantas (UPM-INIA); Universidad Politécnica de Madrid; Escuela Técnica Superior de Ingenieros 
Agrónomos; Campus de Montegancedo; Madrid, Spain
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cellularization process that begins in the 
micropylar region and during seed devel-
opment spreads to the central and chala-
zal regions.2
Seed germination is initiated with water 
uptake by the dry seed and terminates 
with the elongation and emergence of the 
embryonic axis. Physically, germination 
in Arabidopsis is a two-stage sequential 
process, where testa rupture is followed 
by endosperm rupture. Following rup-
ture of the micropylar endosperm (ME) 
by the elongating radicle, germination is 
completed.1,3 This physiological process is 
tightly controlled by environmental con-
ditions, as well as, by the developmental 
program of the seed, in which abscisic 
acid (ABA) and gibberellins (GA) are 
some of the main hormones involved. 
Ethylene (ET), nitric oxide (NO), aux-
ins, etc., are also part of the complex net-
work of interacting signals that together 
with the transcriptional network involved 
in germination are now being actively 
investigated.3-9
Mannans, Endo-β-mannanases 
and Seed Germination
Two major forces play antagonistic roles 
in radicle emergence: the radicle growth 
potential (i.e., primarily cell elonga-
tion),10 and the mechanical resistance of 
the covering layers which is likely to be 
diminished before radicle protrusion by 
cell-wall (CW) hydrolytic enzymes.1,4,5 
©2011 Landes Bioscience.
Do not distribute.
2 Plant Signaling & Behavior Volume 6 issue 1
will ultimately develop into the vascular 
systems of xylem and phloem. Another 
point to be considered is that all the anno-
tated MAN genes in Arabidopsis have pre-
dicted signal peptides, an indication that 
their corresponding proteins will be prob-
ably exported into the apoplastic space and 
could reach the CWs of the ME. Thus, 
radicle and endosperm MAN synthesis 
will contribute together to the weakening 
of the micropylar zones of the endosperm 
(Fig. 2). Remarkably, hormonal coopera-
tion between the embryo and endosperm 
have been demonstrated in other physi-
ological processes.19,20 As far as we know, 
since the radicle of Arabidopsis is also a 
production site of MAN that can be allo-
cated to the periplasmic space, through 
the secretion pathway, it is reasonable to 
postulate a cooperation between embryo 
and endosperm MAN enzymes in the dis-
mantling of the ME CW thus facilitating 
radicle protrusion. Our work18 would be 
the first data to support such a cross-talk 
regarding the synthesis of MAN upon 
germination.
References
1. Nonogaki H, Bassel GW, Bewley JD. Germination—
still a mystery. Plant Sci 2010; In press.
2. Linkies A, Graeber K, Knight Ch, Leubner-Metzger 
G. The evolution of seeds. New Phytol 2010; 
186:817-31.
3. Liu PP, Koizuka N, Homrichhausen TM, Hewitt JR, 
Martin RC, Nonogaki H. Large-scale screening of 
Arabidopsis enhancer-trap lines for seed germination-
associated genes. Plant J 2005; 41:936-44.
4. Finch-Savage WE, Leubner-Metzger G. Seed dor-
mancy and the control of germination. New Phytol 
2006; 171:501-23.
5. Holdsworth MJ, Bentsink L, Soppe WJ. Molecular 
networks regulating Arabidopsis seed maturation, 
after-ripening, dormancy and germination. New 
Phytol 2008; 179:33-54.
6. Rodríguez-Gacio MC, Matilla-Vázquez MA, Matilla 
AJ. Seed dormancy and ABA signaling. The break-
through goes on. Plant Signal Behav 2009; 4:1035-
48.
7. Matilla AJ, Matilla-Vázquez MA. Involvement of eth-
ylene in seed physiology. Plant Sci 2008; 175:87-97.
8. Vicente-Carbajosa JV, Carbonero P. Seed matura-
tion: developing an intrusive phase to accomplish a 
quiescent state. Int J Dev Biol 2005; 49:645-51.
9. Linkies A, Müller K, Morris K, Turecková V, Wenk 
M, Cadman CSC, et al. Ethylene interacts with 
abscisic acid to regulate endosperm rupture during 
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10. Sliwinska E, Bassel GW, Bewley JD. Germination 
of Arabidopsis thaliana seeds is not completed as a 
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60:3587-94.
Is there a Cross-talk between 
Radicle and ME in Relation to 
Endo-β-mannanases during Seed 
Germination?
In our recent study with T-DNA insertion 
mutants in AtMAN genes, we concluded 
that the expression of AtMAN5, AtMAN6 
and AtMAN7 in imbibed seeds is impor-
tant for A. thaliana germination, probably 
by decreasing the mechanical resistance of 
the ME, thus facilitating the radicle emer-
gence.18 Germination time course is nega-
tively affected in knock-out (KO) mutants 
of AtMAN7 and AtMAN5 and strongly 
inhibited in KO AtMAN6 (Fig. 1). In 
parallel, we have detected transcripts of 
AtMAN5, AtMAN6 and AtMAN7 genes 
not only in the ME but also in the radicle, 
thus not ruling out the possibility that 
the AtMAN gene products could be also 
involved in the radicle cell growth through 
a transglycosylase activity. This conclu-
sion is a consequence of the fact that the 
role of MAN as a hydrolase may not be the 
dominant role in CW of the radicle itself, 
where mannans are only minor, although 
important components.12 MAN expres-
sion is high in the vascular elements of the 
radicle, probably indicating a role in emp-
tying the nascent conducting vessels that 
In order for the radicle apex to emerge, 
the growth potential must overcome the 
mechanical resistance of the endosperm. 
ABA inhibits endosperm rupture, but 
not testa rupture, and interacts with ET 
during Arabidopsis and Lepidium germi-
nation.9,11 Since the weakening of endo-
sperm CW is an important factor for 
radicle protrusion in seeds of Arabidopsis, 
it is of great interest to study the genes 
involved in the dismantling of the endo-
sperm CW structure, which, unlike radi-
cle CW, is rich in mannans,12 conferring 
to this tissue a remarkable mechanical 
resistance. Endo-β-mannanase (MAN) 
activity has been reported in seeds of 
several endospermic species before and 
following radicle emergence.13 However, 
although the degradation of mannans 
is initiated by MAN,1,14 the role of this 
enzyme in endosperm weakening is still 
controversial, to the point that some 
authors maintain that MAN is not 
involved, while others conclude that 
MAN is the main enzyme component of 
the CW dismantling process.5,15,16 Taken 
together all these observations, the con-
sensus appears to be that while MAN is 
required for endosperm weakening, itself 
is not enough to allow completion of 
germination.5
Figure 1. Germination time course of Arabidopsis thaliana Wt and t-dna insertion mutant seeds. 
Wt: closed circles (●; t50 = 25); K.O. man7 (GaBi_747H02): Opened triangles (Δ; t50 = 34.4); K.O. 
man5 (GaBi_707G06): Opened circles (○; t50 = 37.5); K.O. man6 (Salk_122701): closed triangles (▼; 
t50 = 48). data are means ± standard error (Se) of three independent experiments.
©2011 Landes Bioscience.
Do not distribute.
www.landesbioscience.com Plant Signaling & Behavior 3
11. Müller K, Tintelnot S, Leubner-Metzger G. 
Endosperm-limited Brassicaceae seed germination: 
abscisic acid inhibits embryo-induced endosperm 
weakening of Lepidium sativum (cress) and endo-
sperm rupture of cress and Arabidopsis thaliana. Plant 
Cell Physiol 2006; 47:864-77.
12. Marcus SE, Blake AW, Benians TAS, Lee KJD, 
Poyser C, et al. Restricted access of proteins to man-
nan polysaccharides in intact plant cell walls. Plant J 
2010; 64:191-203.
13. Yuan JS, Yang X, Lai J, Lin H, Cheng ZM, Nonogaki 
N, et al. The endo-β-mannanase gene families in 
Arabidopsis, rice and poplar. Funct Integr Genomics 
2007; 7:1-16.
14. Moreira LRS, Filho EXF. An overview of mannan 
structure and mannan-degrading enzyme systems. 
Appl Microbiol Biotechnol 2008; 79:165-78.
15. Gong XM, Bassel GW, Wang A, Greewood JS, 
Bewley JD. The emergence of embryos from hard 
seeds is related to the structure of the cell walls 
of the micropylar endosperm, and not to endo-β-
mannanase activity. Ann Bot 2005; 96:1-9.
Figure 2. Proposed model of man enzyme traffic in the germinating seed. man proteins with their signal peptides (SP; ●) are secreted ■ to the apo-
plastic space where they become active after proteolysis of the SP (*). root (rt), micropylar endosperm (me) and seed coat (Sc).
16. Nonogaki H, Chen F, Bradford KJ. Mechanisms 
and genes involved in germination “senso stricto”. In: 
Bradford K, Nonogaki H (Eds.) Seed development, 
dormancy and germination. Blackwell, Oxford 2007; 
264-304.
17. Schröder R, Atkinson RG, Redgwell RJ. 
Re-interpreting the role of endo-β-mannanases as 
mannan endotransglycosylase/hydrolases in the plant 
cell wall. Ann Bot 2009; 104:197-204.
18. Iglesias-Fernández R, Rodríguez-Gacio MC, 
Barrero-Sicilia C, Carbonero P, Matilla AJ. Three 
endo-β-mannanase genes expressed in the micropylar 
endosperm and in the radicle influence germination 
of Arabidopsis thaliana seeds. Planta 2010; In press.
19. Arana MV, de Miguel LC, Sánchez RA. A phy-
tochrome dependent embryonic factor modulates 
gibberellin responses in the embryo and micropylar 
endosperm of Datura ferox seeds. Planta 2006; 
223:847-57.
20. Arana MV, Burgin MJ, de Miguel LC, Sánchez RA. 
The very-low-fluence and high-irradiance respons-
es of the phytochromes have antagonistic effects 
on germination, mannan-degrading activities and 
DfGA3ox transcript levels in Datura ferox seeds. J Exp 
Bot 2007; 58:3997-4004.


Molecular analysis of endo-β-mannanase genes upon seed imbibition suggest a cross-talk between radicle and micropylar endosperm during germination of Arabidopsis thaliana

Molecular analysis of endo-β-mannanase genes upon seed imbibition suggest a cross-talk between radicle and micropylar endosperm during germination of Arabidopsis thaliana

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