75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

179 Investigation Of The Connection Between Leaf Polarity And Meristem Formation Through
Enhancers Of pinhead
Debbie Alexander, Karyn Lynn Newman, M. Kathryn Barton
Carnegie Institution of Washington
Several lines of evidence indicate that the adaxial leaf domain possesses a unique competence to promote shoot apical meristem
formation and maintenance. The Arabidopsis PINHEAD (PNH) gene likely plays a role in this process as PNH is expressed in
adaxial domains and loss of function pnh mutants fail to maintain the indeterminacy of shoot meristems. Furthermore, PNH acts
redundantly with ARGONAUTE1 (AGO1) to control organ polarity. PNH and AGO1 are closely related members of the Argonaute
family of proteins. Argonaute proteins are core components of RNA-induced silencing complexes (RISC) which act in small RNAdirected
silencing of target mRNAs. While it is likely that PNH also interacts with small RNAs, it is unclear whether the output
of this interaction would be translational repression, cleavage of target mRNAs, methylation of target genes, or other processes.
To investigate the role of PNH in leaf polarity and meristem formation, we screened for EMS generated enhancers
of the pnh-2 phenotype. To maximise the likelihood of isolating genes involved in the same pathway as PNH,
plants were screened for the narrow petal phenotype characteristic of ago/+;pnh/pnh double mutant plants.
The initial screen produced 34 enhancer of pinhead (enp) lines. Five of these lines do not exhibit a mutant phenotype in the
absence of pnh and were selected for further analysis. The lines can be grouped into 3 phenotypic classes: 1) narrow petals, 2)
splayed flowers and 3) ectopic floral organs. Within the recessive class 1 enhancers, double enp1/enp1;pnh/pnh and enp2 pnh/
enp2 pnh mutant plants closely resemble ago/+;pnh/pnh mutants, with individual lines differing in phenotypic severity. When
doubly mutant with pnh, class 2 enhancers, enp3 and enp4 confer distinctive phenotypes such as splayed floral organs and green
and white coloured sepals. The class 3, semi-dominant enhancer, enp5, is the only enp mutation to modify both vegetative and
reproductive pnh mutant phenotypes. The vegetative SAM of double pnh enp5/pnh enp5 mutants never terminates in a ‘pin’
structure and ectopic floral meristems develop in longitudinal rows along the fasciated inflorescence stem. Preliminary data
suggests that enp5 is a novel allele of REVOLUTA, an HD-ZIP III family member involved in organ polarity and meristem
formation. REV expression is controlled by microRNA regulation, raising the intriguing possibility that PNH influences organ
polarity through the control of REV expression. Future experiments will test this possibility and determine the identity of the
other enp genes.
180 Elucidating the Role of Basic Helix-Loop-Helix (bHLH) Transcription Factors in Stomatal
Development
Naomi Bogenschutz, Lynn Jo Pillitteri, Dan Sloan, Keiko Torii
Department of Biology, University of Washington, Seattle, Washington 98195
Stomata consist of a pair of guard cells, which operate as a turgor-driven valve required for efficient gas and
water exchange between a plant and its environment. Stomatal formation begins when an undifferentiated epidermal
cell called the meristemoid mother cell (MMC) divides asymmetrically. The smaller daughter cell is called a
meristemoid, which renews itself through several rounds of divisions before differentiating into a guard mother cell
(GMC). The GMC then divides symmetrically, producing two guard cells surrounding a pore to form a mature stoma.
Several cell-signaling molecules have been shown to regulate early steps in stomatal development, including the production
of MMCs and the subsequent frequency or orientation of asymmetric divisions. Loss-of-function mutations in TOO
MANY MOUTHS (TMM) [1], STOMATAL DENSITY AND DISTRIBUTION (SDD) [2], YODA (YDA) [3], and the
ERECTA (ER)-family [4] cause stomata to form next to each other or in clusters, suggesting that these genes transmit cell
position and orientation signals. Loss-of-function mutations in genes controlling later steps in stomatal differentiation,
such as FOUR LIPS (FLP) [5] and FAMA [3], cause repetitive GMC divisions resulting in stacks of several guard cells,
demonstrating their roles in controlling guard cell proliferation. In contrast, no genes have been found that regulate the
commitment of a meristemoid to a GMC. By visually screening an ethyl methanesulfonate-mutagenized population of
Arabidopsis, we have identified a novel gene, MUTE, that acts as a master regulator for terminal differentiation of the
meristemoid to a GMC. MUTE encodes a basic helix-loop-helix (bHLH) transcription factor, where loss-of-function
produces a no stomata or “no mouths” phenotype. Unlike wild-type meristemoids that differentiate after 1 to 3 divisions,
mute meristemoids divide asymmetrically 3 to 6 times, producing a rosette pattern with an arrested meristemoid at the
center. We also determined that a closely related paralog of MUTE is a critical regulator of stomatal development by
controlling the initial asymmetric division of the MMC, the first step in the stomatal formation pathway. Characterization
of MUTE and its place in the stomata gene regulatory network will be presented.
References: [1] Berger and Altmann (2000) Genes Dev. 14, 1119. [2] Nadeau and Sack (2002) Science 296, 1697. [3] Bergmann et al. (2004)
Science 304, 1494. [4] Shpak et al. (2005) Science 309, 290. [5] Lai et al. (2005) Plant Cell 17, 2754.