Plant cells have a variety of shapes crucial for their functions, yet the mechanisms that generate these shapes are poorly understood. Genetic dissection of the trichome (plant hair) branching pathway in Arabidopsis, has uncovered
mechanisms and identified genes that control plant cell morphogenesis. The recent identification of one of these genes, ZWICHEL(ZWI), as a novel member of the kinesin superfamily of microtubule motors provides a starting point for the analysis of the plant cytoskeleton’s role in a specific morphogenetic event.
Addresses
Department of Biological Sciences and Coalition for BioMolecular Products, 301 Biology, University of Alabama, Tuscaloosa, AL 35487-0344, USA; e-mail: [email protected]
Current Opinion in Plant Biology1998, 1:520–524 http://biomednet.com/elecref/1369526600100520 © Current Biology Ltd ISSN 1369-5266
Abbreviations
CaMV cauliflower mosaic virus
KCBP kinesin-like calmodulin-binding protein
KRP kinesin-related protein
Introduction
Arabidopsistrichomes are a well-established system used to address questions concerning cell shape and cell expan-sion [1–5]. The trichome’s distinctive shape (Figure 1) allows the easy identification of mutants with alterations in
trichome form, including changes in size, branch number, and overall morphology.
The first systematic screen for trichome mutations identi-fied 21 loci that control different aspects of trichome differentiation including trichome cell fate specification, trichome morphology, trichome cell size, and trichome cell wall maturation [5]. Since then, many additional mutations affecting trichome morphology have been identified [6••,7]
(DG Oppenheimer, unpublished data, D Larkin and DG Oppenheimer, unpublished data)
Trichome morphology mutations can be grouped into classes on the basis of their effect upon trichome shape [5,6••,7••,8–10]; several mutations affect more than one of
the trichome differentiation processes (Table 1). The processes affected by each of the mutations suggest points of control of the trichome developmental program. For example, trichome branch number can be altered by muta-tion — either increased or decreased — independently of trichome maturation. This suggests that both positive and negative regulators control trichome branch number, and that trichome branch number is regulated independently of trichome maturation.
Wild-type trichome morphogenesis in
Arabidopsis
The trichome is the first cell type to differentiate on the epidermis of leaf primordia (Figure 1). Protodermal cells
Genetics of plant cell shape
David G Oppenheimer
Figure 1
Current Opinion in Plant Biology
(a) (b) (c)
Scanning election micrographs of trichomes on Arabidopsisplants.
(a)Developing second leaf pair of a wild-type plant showing trichomes in all stages of development. Small arrow indicates a developing trichome undergoing branch initiation. Large arrow indicates a mature
committed to the trichome cell fate cease cell division, but continue to replicate their DNA; nuclear enlargement is the first observable sign that trichome differentiation has started [5]. After approximately three rounds of endorepli-cation, the incipient trichome expands out of the leaf primordium. An additional round of endoreplication occurs, and the trichome initiates two or three branches in quick succession. Generally, the first branch is oriented toward the tip of the leaf primordium, and the second branch is oriented at a right angle relative to the first branch. Following branch initiation, the nucleus migrates from a central position in the stalk to the base of the sec-ond branch point. The trichome continues to expand until it reaches a height of 200-300µm and a base diameter of approximately 50µm. During the final maturation phase, the trichome cell wall thickens, papillae develop on the surface of the trichome, and rectangular-shaped cells develop around the base of the trichome.
The role of
ZWI in trichome morphogenesis
Mutations in the zwigene cause a failure of the trichomes to branch properly; the result is a two-branched trichome with a greatly shortened stalk [5,6••,11•]. The ZWI genewas cloned using a T-DNA tagged zwi allele [11•], and
found to encode a kinesin-related protein (KRP) that was previously identified by Reddy and coworkers in a screen for calmodulin-binding proteins [12]. Its ability to bind Ca2+/calmodulin defines theZWI product, named KCBP for kinesin-like calmodulin-binding protein, as a novel member of the kinesin superfamily of microtubule motor proteins. Microtubule motor proteins use energy from the hydrolysis of ATP to move ‘cargo’ along microtubule ‘tracks’ within a cell. A typical KRP consists of three parts: the motor domain which binds ATP and microtubules, a tail domain which is thought to bind to the cargo, and a stalk domain which connects the motor and tail domains and par-ticipates in homodimerization. Different members of the KRP family show little homology to each other outside of the conserved motor domain. The identification of theZWI product presents the opportunity to biochemically and genetically dissect the function of a cytoskeletal protein that has a specific effect on the shape of a single cell type.
What is the role of KCBP in trichome branching? Trichome branching can be thought of as a series of local-ized, cell wall expansion events. The cortical array is thought to control the direction of cell expansion by con-trolling the orientation of newly synthesized cellulose microfibrils in the cell wall [13,14]. In this context, a change in the direction of cell expansion (i.e., trichome branching) is preceded by a reorganization of the cortical array at the site of future expansion. This reordering of the cortical microtubules could be accomplished by the cell in either of two ways [15,16]. First, the microtubules in the cortical array could depolymerize and then repolymerize in the orientation needed for the change in the direction of expansion. This would require a mechanism that targets microtubule organizing centers to the correct intracellular
locations prior to microtubule repolymerization. Alternatively, the microtubules could be moved into their positions by a motor protein after their polymerization. This is attractive in light of the requirement of a motor protein (KCBP) for trichome branching.
One hypothesis of the role of KCBP during trichome branch formation is that KCBP participates in the reorga-nization of the cortical array microtubules prior to branch initiation. This hypothesis is supported by the following observations: KCBP has been localized to microtubule arrays (the preprophase band, the spindle, and the phrag-moplast) in dividing cultured plant cells [17•]. KCBP is a
member of the C-class of KRPs that function as minus-end directed motors; the motor domain of this subclass of KRPs is located at the carboxy-terminus of the protein and moves the KRP toward the minus end of microtubules. C-type KRPs generally participate in microtubule move-ments [18,19,20•]. In addition, Ca2+/calmodulin can affect
the stability of the cortical array [21], and can inhibit the binding of KCBP to microtubules in vitro[19,22••]. These Table 1
Mutations that affect trichome development in Arabidopsis.
Effect of mutation on Name of mutant References trichome development
Increase/decrease cell size glabra3 (gl3) [5,6••]
glabra2 (gl2) [8]
gumby(gmb) *
kaktus(kak) [5]
noeck(nok) [5,6••]
small trichomes1 (sml1) †
triptychon (try) [5,6••] Decrease branch number angustafolia(an) [10]
furca1(frc1) ‡
furca2(frc2) ‡
furca3(frc3) ‡
furca4(frc4) ‡
gl3 [5,6••]
stachel(sta) [5,6••]
stichel(sti) [5,6••]
zwichel(zwi) [5,6••] Increase branch number kak [5,6••]
nok [5,6••]
suppressor of zwichel2 (suz2) §
try [5,6••]
Distort overall morphology alien(ali) [5,6••]
crooked(crk) [5,6••]
distorted1(dis1) [5]
distorted2(dis2) [5]
gnarled(grl) [5,6••]
spirrig(spi) [5,6••]
wurm(wrm) [5,6••] Block maturation chablis(cha) [5]
chardonnay(cdo) [5]
nok [5,6••]
retsina(rts) [5] *TA Venezia, JC Larkin and DG Oppenheimer, unpublished data;
†DG Oppenheimer, unpublished data; ‡D Luo and DG Oppenheimer,
observations are consistent with a role of KCBP in the organization of the trichome cortical array microtubules. The intracellular location of KCBP in trichomes is not yet known, but immunolocalization experiments are currently underway in the author’s laboratory. If KCBP functions in reorganization of the trichome cortical microtubules prior to branching, we may expect to see localization of KCBP to the cortical array in developing trichomes.
The genetic analysis of ZWIhas also provided insight into its role in branch initiation. The zwimutation results in tri-chomes with fewer branches and a shortened stalk [5,6••,11•]. Although the expression of theZWI gene
has been detected in many Arabidopsistissues, strong zwi mutants display only a trichome defect [5,6••,11•,12]. This
result suggests thatZWI function in tissues other than tri-chomes is redundant.
Results from a genetic analysis of trichome branching sug-gested thatZWI was required for branching competence of the trichome cell [6••]. Results from a recent screen for
suppressors of the strong zwi-3 mutation [11•], however,
suggest that ZWI plays a more direct role in trichome branching (S Krishnakumar and DG Oppenheimer, unpublished data). One of the suppressors isolated in the screen, suz2, rescues the branch defect of the zwi-3 muta-tion; although no three-branched trichomes are found on zwi-3 plants, more than one-half of the trichomes on suz2 zwi-3 double mutants have three branches. In addition, trichomes onsuz2 single mutants have more branches than wild-type trichomes, indicating that SUZ2is a nega-tive regulator of trichome branching (S Krishnakumar and DG Oppenheimer, unpublished data). The ability of the suz2mutation to rescue trichome branching in zwi-3 suz2 double mutants suggests that the zwi mutation does not compromise branching competence.
The relationship between trichome cell size
and trichome shape
Upon entering the trichome differentiation pathway, a pro-todermal cell ceases cell division but continues to replicate its DNA. Prior to branching, the nascent trichome com-pletes three rounds of endoreplication concurrent with cell growth. Endoreplication is required for growth and branch-ing of the trichome as suggested by the genetic analysis of two mutants: glabra3 (gl3) and triptychon (try) [6••]. In gl3
mutants, the third round of endoreplication is blocked, resulting in smaller trichomes with fewer branches than in wild-type plants. Mutations in gl3are epistatic to other tri-chome branch mutations. These results suggest that a minimum cell size (or a minimum level of endoreplication) is needed for trichome branching [5,6••]. Mutations in
theTRY gene result in extra rounds of endoreplication, increased final cell size, and supernumerary branches. This correlation suggests that the result of the increased cell size is additional trichome branches. Taken together, the analy-ses of both gl3 and try mutants suggest that trichome branching is regulated by cell size and/or cell growth [5,6••].
Recent analyses of trichome ploidy levels in plants expressing the GL1gene driven by the constitutive cauli-flower mosaic virus (CaMV) 35S promoter (35S::GL1 plants), however, suggest that the relationship between ploidy level and branch number may be more complex. Trichomes on 35S::GL1 plants are phenotypically wild-type in size and branch number [23,24], but have the increased level of endoreplication of trytrichomes [25].
In addition, trichomes on the newly discovered gmbmutants are smaller than wild-type trichomes, but have the same number of branches as wild-type (TA Venezia, JC Larkin, and DG Oppenheimer, unpublished data). Preliminary measurements of ploidy levels indicate that trichome endoreplication is reduced in gmb mutants (TA Venezia, JC Larkin, and DG Oppenheimer, unpublished data). The isolation of additional mutations that affect ploidy levels, and the assessment of their effect on trichome branch num-ber and cell size will help clarify the relationship between endoreplication and trichome morphology.
A new model of the regulation of trichome
branch number
Genetic analysis of trichome branch formation has identi-fied seven genes that regulate trichome branching [6••].
The careful characterization of the phenotypes of both sin-gle and double mutants resulted in a model of the trichome branching pathway. Reviews of this work have been published recently [9,10]; therefore, this section of the review will focus on the extension of the published model using results from the genetic analysis of new tri-chome branch mutants.
Four new genes that control trichome branch number have been identified (D Luo and DG Oppenheimer, unpublished data). Recessive mutations in these genes result in trichomes with two branches instead of three (the mutants were named furca[frc] which is Latin for a two-pronged fork). All pairwise combinations of double mutants were constructed between the frcmutants and all the other trichome branch number mutants. In addition, all possible pairwise combinations of frc double mutants were constructed. The double mutant phenotypes were interpreted as follows: if the double mutant phenotype was the same as the phenotype of either parent, then the two genes were placed on the same pathway. If the dou-ble mutant did not look like either parent, or had a novel or additive phenotype, then the two genes were placed on separate pathways.
The results of these double mutant analyses led to a revised model of the trichome branch initiation pathway (Figure 2). As in the original model of trichome branching regulation [6••], the revised model proposes that both
In the revised model, four independent pathways con-tribute to branch initiation. The number of branches initiated depends on the levels of four regulators: AN, ZWI, FRC1, and FRC3. For example, increased expression ofAN andZWI (due to loss of negative regulation by NOK), leads to an increase in trichome branches. The function of the four regulators is redundant: frc1mutations are rescued by mutations inNOK orTRY (by the increase in expression of AN and ZWI). Blocking any one of these four pathways results in trichomes that initiate only one branch. If two pathways are blocked, no branches are initiated. Currently, this model is consistent with the phenotypes of all the known pairwise double mutants (DG Oppenheimer et al., unpublished data).
The revised model differs fundamentally from the model previously published by Folkers et al.[6••], where it was
proposed that primary and secondary branching are genet-ically distinct, with STAregulating primary branching, and AN regulating secondary branching. Trichomes on an mutants make only the primary branch (which is oriented parallel to the midvein of the leaf), but cannot make sec-ondary branches. Conversely, stamutants lack the primary branch, yet secondary branching is unaffected. Thus sta mutant trichomes have two branches that are oriented at random with respect to the midvein of the leaf. The an sta double mutants do not initiate either primary or secondary branches and thus remain unbranched. In the revised model, primary and secondary branching are not genetical-ly distinguishable, as illustrated by the following observations. The trichomes on frc1, frc2, and frc3mutants are similar to those on anmutants, both with respect to the
orientation of the trichome relative to the midvein (proxi-modistal axis) of the leaf, and with respect to branch number. The trichomes on frc4mutants resemble those on sta mutants (D Luo and DG Oppenheimer, unpublished data). Using the original branch initiation model proposed by Folkers et al.[6••], FRC1, FRC2, and FRC3 might be
placed in the secondary branch initiation pathway, and FRC4in the primary branch initiation pathway. The origi-nal model predicts that double mutant combinations of an, frc1, frc2, and frc3 should have phenotypes no different from each single mutant. In addition, the original model predicts that double mutant combinations of stawith frc1, frc2, and frc3 should have unbranched trichome pheno-types. When pairwise double mutants were constructed, however, all the double mutant combinations of an, frc1, frc3, and frc4produced plants with unbranched trichomes. In addition, most trichomes on double mutants between sta and frc1, frc2, or frc3 have two branches (D Luo and DG Oppenheimer, unpublished data). Analysis of the new frcmutants, therefore, suggests that there is not a genetic distinction between primary and secondary branching.
Conclusions
The past year has been an exciting one for those working on plant cell shape; however, the cloning of only three of the 35 genes involved in trichome differentiation has been reported [8,11•,26]. Clearly, to understand the mechanisms
controlling cell morphogenesis at the molecular level, the products of many more of these genes must be identified. Fortunately, the number of T-DNA and transposon muta-genized lines is growing rapidly — thus increasing the chance of identifying additional tagged trichome mutants.
Figure 2
Genetic control of trichome branch initiation.
(a)The original model of trichome branch number control [6••]. Blunt lines indicate negative regulation. Arrows indicate positive regulation. Dashed lines indicate a qualitative requirement; solid lines indicate quantitative regulation. (b)A revised model of the trichome branch initiation pathway. The model illustrates additive and epistatic genetic relationships among genes that are currently known to control trichome branch number. Positive regulation is indicated by arrows. Dashed arrows indicate a weaker regulation than solid arrows. Negative regulation is indicated by lines terminated by a perpendicular bar. Four independent pathways control branch initiation. Branch number is controlled by four regulators:
FRC1,AN, ZWI, and FRC3. The levels of expression of the four regulators determines the number of branches. Loss of function of any one of the four regulators by mutation results in a reduction in trichome branch number. Loss of function of one of the four regulators can be rescued by an increase in the level of another of the regulators. Mutations in the following genes lead to a
decrease in trichome branch number: FRC1,
FRC2, FRC3, FRC4, AN, ZWI, STA, and
GL3. Mutations in NOK, SUZ2, or TRYcause an increase in trichome branch number. The
ploidy level of the trichome is decreased in gl3
mutants and increased in trymutants. Branch initiation Primary branch formation
Secondary branch formation
Endo-replication
FRC1
FRC2 GL3
GL3
TRY TRY
FRC4 STA
STA STI NOK
AN
SUZ2
NOK AN
ZW1
FRC3
Current Opinion in Plant Biology Cell growth/size
(endoreplication) (a)
In addition, the Arabidopsisgenome sequence will contin-ue to be an essential tool for map-based cloning strategies. At least seven of the currently known trichome mutations are each represented by only a single allele; thus, it is unlikely that the Arabidopsis genome has been saturated for trichome mutations. In the next few years, we can look forward to the discovery of more genes involved in tri-chome morphogenesis, and the identification of more gene products involved in the control of trichome cell shape.
Acknowledgements
I would like to thank John Larkin, Danlin Luo, Janis O’Donnell, and Ed Stephenson for helpful discussions, Mary Pollock, Ed Stephenson, and Sujatha Krishnakumar for comments on the manuscript, and Jolanta Nunley for the scanning electron micrographs in Figure 1. I apologize to those colleagues working in the field whose work was not mentioned due to space limitations. I also gratefully acknowledge financial support from the National Institutes of Health (R29-GM 53703).
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