ChangSuDepartmentof Bioscience, Institute of Biotechnology, University of HelsinkiWood is one of the most sustainable and renewable resources,which can provide materials, energy and environmental benefits for humans.Basically, wood is the secondary xylem of woody plants. In recent years, manyregulatory mechanisms controlling wood formation had been revealed based onplant hormones. It is also apparent that plant hormones rarely work alone, butrather interact and crosstalk. Here, the most significant and recent researchresults are summarized to help us understand plant hormones regulation and crosstalkduringwood formation.
Keyword: Cambium, Xylem, Plant hormone IntroductionAnatomically, the trucks of trees contain pith, xylem, cambium,phloem and bark, from the most inner side to the most outer side. The cambiumis a meristematic tissue layer that provides undifferentiated cellscontinuously. Cambial cells proliferate and xylem cells are formed. Thesecondary xylem is also called as wood. Wood serves two main function, supportingthe tree physically, meanwhile conducting water and nutrients for the needs ofplant growth and defense. The secondary xylem cell walls are highly structuredand well assembled by four major compound: polysaccharides (cellulose,hemicelluloses), lignin, organic extractives and inorganic elements (Higuchi,1997). Xylem (hardwood) contains five cell type: vessel element, tracheid, raycell, parenchyma cell and fiber. Wood (secondary xylem)is manufactured by a succession of five major steps, including cell division,cell expansion, cell wall thickening, programmed cell death, and heartwoodformation.
Many regulatory mechanism control the wood formation process,including DNA modification, transcriptional regulation, post-transcriptionalmodification, post-translational regulation. The most well-known studies arebased on transcriptional regulation, which is highly regulated by plant hormonesand transcription factors. AUXINCambium is a key factor of wood (secondary xylem) formation,because cambial cells proliferate and provide source for xylem differentiation.Auxin has been considered as a key regulator of cambium activity, since thecambial growth was re-activated of the decapitated plant has been observed afterthe classical hormones treatment (1). Further evidence is that the gradient auxinconcentration peaked in cambium dividing cells and decreased in the xylem (2, 3) has been detected inpine and aspen wood. One well-established hypothesis is the polar auxintransport (PAT) keeps a directional auxin gradient (4).
The directional transport assisted by PINproteins. The directional gradient results in cambial cells maintenance anddifferentiation. Genetic screen for defective vascular mutants has successfullyidentified some Arabidopsis mutants (5-7), for instance: arf5, axr5 and iaa12. These mutations happened in auxin signaling pathway,resulted in having defective vascular and consequently defective vasculardifferentiation. van3 mutation hadthe fragmented vascular phenotype, associated with disrupted vascular networkand decay of PIN2 expression (8). Triple loss-of-function kan1;kan2;kan4 embryo, which defected the peripheral–central axis, changedthe expression of PIN1 and the polar localization (9).
All of this suggest auxinand auxin transport (assisted by PIN proteins) control vascular pattern. InPopulous, the anticlinal divisions only happens in the phloem side of cambium (10). But in the IAA(auxin signaling gene) overexpressing trees, the anticlinal divisions covered awider zone.
In the transgenic tree overexpressing the repressor of the IAAgene, the transgenic trees reduced auxin responsiveness, then reducedanticlinal divisions. The last, this tree reduced wood formation and formedthinner stems, compared to wild type (11). These resultsindicats auxin regulates wood formation by regulating cambium activity. CYTOKININCytokinins (CK) are central regulators of cambium activity and regulatexylem formation. IPT (ATP/ADP isopentenyltransferase) genes are biosynthesisgenes in CK pathway. Quadruple mutant ipt1;3;5;7had thinner inflorescence stem and roots compared to wild type Arabidopsis. Theweak phenotype mutant was rescued by 2000ng/ml CK treatment. But 200ng/ml CKtreatment was only able to partially rescue the phenotype (12).
CRE1/WOL is the first identified plant CK receptor(13). The root of recessive wol mutant resulted in fewer cells in vascular, and all these cellsdifferentiated to xylem, rather than phloem (14). CRE1/WOL and two additional members, AHK2and AHK3, activate a phosphorylation cascade characteristic of prokaryotictwo-component hybrid molecule signaling, acting as CK receptors.
Downstreamfunctional response regulators (RRs), which are involved in CK signaling, aredivided into three groups, two of which (type A and type B). Type B ARRs bindto DNAand initiate transcription of downstream targets afterphosphorylation (15). Type A ARRs are considered as inhibitors ofCK signaling (16). Triple loss-of-function arr1;arr10;arr12 mutant, which contained mutation in type-B ARRs, resulted in wol-like phenotype.
These result explained CK regulates cambiumactivity and xylem formation. Overexpressing a cytokinin catabolic gene, CYTOKININ OXIDASE 2, in Populous, resulted in a reduction ofbiologically active CK. The apical and radial growth were compromised in thistransgenic tree. But the radical growth was more comprised (17). To understand moreabout how CK affect cambium activity and wood formation in trees, the overexpressingIPT7 transgenic tree was analyzed byImmanen et al (18). The overexpressing IPT7 tree significantly increased stemdiameter and lignocellulosic biomass. Interestingly, overexpressing IPT7 not only increased CKconcentration, but also auxin concentration in stem (18).
These evidenceconfirmed the CK positive regulates cambium activity in trees. The CK signalingconnects to the auxin signaling rather than works alone.Several genes have been found that affect xylem formation(reviewed by Fukuda (19)). By the invitro xylem vessel element formation system, VND6 and VND7, two genesfrom the VASCULAR-RELATED NAC-DOMAINfamily, have been showed specified xylem identity in both Arabidopsis andPopulous.VND6 and VND7 induce trans-differentiation ofvarious cells into metaxylem- and protoxylem-like vessel elements, respectively(20). Further studiesconfirmed VND6 and VND7 regulate genes involved in a widerange of processes in xylem vessel differentiation (21, 22).
To understand howplant hormones modulate this two genes in the in vitro xylem vessel element formation system. The plant cellculture had been treated with auxin, cytokinin, and brassinosteroids. Auxin andcytokinin were required to obtain ”significant” expression. VND6 and VND7 had higher expressionlevel after the treatment with all three hormones, which might worksynergistically (20). In this cell culturesystem, brassinosteroids levels increases prior. It meansbrassinosteriods is necessary in the process of xylem differentiation (23).
BrassinosteriodsAs mentioned before, brassinosteriods (BR) might worksynergistically with auxin and CK to regulate xylem formation by up-regulationof VND6 and VND7 (20), as well as HD-ZIPIII transcription factors (TFs) (24). These TFs have the same HD-ZIPIII DNAbinding domain. They are involved in formation and differentiation of xylem.Evidence is supported by the mutations in HD-ZIPIIITFs (rev-10D, phb-d, phv-d),which resulted in phloem surrounding by xylem. In the in vitro xylem vessel element formation system, transcripts of HD-ZIPIII genes accumulate when thexylem differentiation initiated (24).
It also showed that auxin induced the AtHB8 (one of the HDZIPIIIs) expression,and auxin genes were down-regulated by the ATHB8(25). GibberellinGibberellin (GA)promotes cambium activity and xylem development. Auxin, CK and GA spread acrossthe cambium to the developing phloem and xylem. Among them, Auxin peaks in thecambial region; CK peaks in the developing phloem; but GA peaks in thedeveloping xylem (18, 26). Only auxinapplication to the decapitated plant stimulated the cambial division and xylemproduction. But only GA application kept cambium derivatives in parenchymaticstatus (27). Application both auxin and GA promotedcambial division and xylem production.
Also the IAA concentration in stemtissue is higher when auxin and GA both were applied than only applied auxin (27). All these hormones treatment gave us the cluethat auxin and GA work synergistically to control wood formation. Thetransgenic populous overexpressing GA biosynthesis grew faster and obtainedmore biomass than wild type. Moreover, overexpressing GA trees produced longerxylem fiber, which is useful for pulping (28).
Furthermore, Björn et al showed GA deficiency reduced auxin transport by vacuolardegradation of PIN proteins, at least PIN2 in this case (29). Correspondently, auxin signaling regulate GAmetabolism gene transcription (30). EthyleneIn addition to auxin,CK, BR, and GA, ethylene is another plant hormone can affect cambialactivities.
Ethylene treatmentresulted in more parenchyma cells from cambium, shorter fibers and vessels inthe xylem compared the control, finally the stimulation of radial growth (31). Ethylene also promotes tension woodformation rather than only a stimulator of cell division in cambium (32). Overexpressingethylene biosynthesis enzyme in Populous provided us xylem-increased transgenictrees. But ethylene insensitive trees produced less tension wood than wild typewhen tilted the intensive tree and wild type tree (32). StrigolactoneStrigolactone (SL) asa newly emerging plant hormone is not well studies as others. Only knowledge weknow about secondary growth is, SL positively regulates cambium activity.Mutations in signaling or biosynthesis resulted in a reduction of cambiumactivity (33).
For example, the max1 mutant, which has mutation in a SL biosynthesis pathway gene,had narrow inflorescence cambium-derived tissue (33). ConclusionWood, the secondaryxylem is differentiated from cambium, which proliferates undifferentiated cellscontinuously. Plant hormones regulate the cambium activity and xylemdifferentiation. Although we have knowledge of some hormonal mechanisms behind woodformation, there are still space we need to fill.