Noritaka Matsumoto

Roles of the Middle Domain-Specific WUSCHEL-RELATED HOMEOBOX Genes in Early Development of Leaves in Arabidopsis

This work proposes that the middle domain, which is distinct from the adaxial (upper) and abaxial (lower) domains, plays a key role in coordinating two important processes in early leaf development, blade outgrowth, and adaxial/abaxial patterning, through the actions of the middle domain–specific WOX genes, PRS and WOX1, in concert with the adaxial- and abaxial-specific genes. During leaf development in flowering plants, adaxial (upper) and abaxial (lower) side–specific genes are responsible for blade outgrowth, which takes places predominantly in the lateral direction, and for margin development as well as differentiation of adaxial and abaxial tissues. However, the underlying mechanisms are poorly understood. Here, we show that two WUSCHEL-RELATED HOMEOBOX (WOX) genes, PRESSED FLOWER (PRS)/WOX3 and WOX1, encoding homeobox transcription factors, act in blade outgrowth and margin development downstream of adaxial/abaxial polarity establishment. The expression of PRS and WOX1 defines a hitherto undescribed middle domain, including two middle mesophyll layers and the margin, as a center that organizes the outgrowth of leaf blades. The expression of PRS and WOX1 is repressed in the abaxial leaf domain by the abaxial-specific transcription factor KANADI. Furthermore, PRS and WOX1 coordinate adaxial/abaxial patterning together with adaxial- and abaxial-specific genes. Our data suggest a model of blade outgrowth and adaxial/abaxial patterning via the middle domain–specific WOX genes in Arabidopsis thaliana leaves.

RABBIT EARS, encoding a SUPERMAN-like zinc finger protein,regulates petal development in Arabidopsis thaliana

Floral organs usually initiate at fixed positions in concentric whorls within a flower. Although it is understood that floral homeotic genes determine the identity of floral organs, the mechanisms of position determination and the development of each organ have not been clearly explained. We isolated a novel mutant, rabbit ears (rbe), with defects in petal development. In rbe, under-developed petals are formed at the correct position in a flower, and the initiation of petal primordia is altered. The rbe mutation affects the second whorl organ shapes independently of the organ identity. RBE encodes a SUPERMAN-like protein and is located in the nucleus, and thus may be a transcription factor. RBE transcripts are expressed in petal primordia and their precursor cells, and disappeared at later stages. When cells that express RBE are ablated genetically, no petal primordia arise. RBE is not expressed in ap1-1 and ptl-1 mutants, indicating that RBE acts downstream of AP1 and PTL genes. These characteristics suggest that RBE is required for the early development of the organ primordia of the second whorl.

Succinic Semialdehyde Dehydrogenase is Involved in the Robust Patterning of Arabidopsis Leaves along the Adaxial–Abaxial Axis

Polarity along the adaxial-abaxial axis of the leaf is essential for leaf development and morphogenesis. One of the genes that encodes a putative transcription factor regulating adaxial-abaxial polarity, FILAMENTOUS FLOWER (FIL), is expressed in the abaxial region of the leaf primordia. However, the molecular mechanisms controlling the polarized expression of FIL remain unclear. Here, we analyzed an enlarged fil expression domain1 (enf1) mutant of Arabidopsis, which forms both abaxialized leaves and adaxialized leaves. The ENF1 gene encodes SUCCINIC SEMIALDEHYDE DEHYDROGENASE (SSADH), which catalyzes the conversion of succinic semialdehyde (SSA) to succinate. The enf1 phenotype was suppressed by an additional mutation in GAMMA-AMINOBUTYRIC ACID AMINOTRANSFERASE1 (GABAT1), which encodes an SSA-producing enzyme, suggesting that SSA or its derivatives is the metabolite responsible for the defect in the adaxial-abaxial axis-dependent gene expression of enf1. In the shoot apical meristem, GABAT1 was expressed in the outermost layer but SSADH was not. Exogenous application of SSA induced adaxial characters on the abaxial side of the newly developed leaves. We suggest that a GABA shunt metabolite, SSA or its close derivatives, is involved in the robust leaf patterning and structure along the adaxial-abaxial axis.

Pattern dynamics in adaxial-abaxial specific gene expression are modulated by a plastid retrograde signal during Arabidopsis thaliana leaf development.

The maintenance and reformation of gene expression domains are the basis for the morphogenic processes of multicellular systems. In a leaf primordium of Arabidopsis thaliana, the expression of FILAMENTOUS FLOWER (FIL) and the activity of the microRNA miR165/166 are specific to the abaxial side. This miR165/166 activity restricts the target gene expression to the adaxial side. The adaxial and abaxial specific gene expressions are crucial for the wide expansion of leaf lamina. The FIL-expression and the miR165/166-free domains are almost mutually exclusive, and they have been considered to be maintained during leaf development. However, we found here that the position of the boundary between the two domains gradually shifts from the adaxial side to the abaxial side. The cell lineage analysis revealed that this boundary shifting was associated with a sequential gene expression switch from the FIL-expressing (miR165/166 active) to the miR165/166-free (non-FIL-expressing) states. Our genetic analyses using the enlarged fil expression domain2 (enf2) mutant and chemical treatment experiments revealed that impairment in the plastid (chloroplast) gene expression machinery retards this boundary shifting and inhibits the lamina expansion. Furthermore, these developmental effects caused by the abnormal plastids were not observed in the genomes uncoupled1 (gun1) mutant background. This study characterizes the dynamic nature of the adaxial-abaxial specification process in leaf primordia and reveals that the dynamic process is affected by the GUN1-dependent retrograde signal in response to the failure of plastid gene expression. These findings advance our understanding on the molecular mechanism linking the plastid function to the leaf morphogenic processes.

VAJ/GFA1/CLO is Involved in the Directional Control of Floral Organ Growth

Flowers assume variant forms of reproductive structures, a phenomenon which may be partially due to the diversity among species in the shape and size of floral organs. However, the organ size and shape of flowers usually remain constant within a species when grown under the same environmental conditions. The molecular and genetic mechanisms that control organ size and shape are largely unknown. We isolated an Arabidopsis mutant, vajra-1 (vaj-1), exhibiting defects in the regulation of floral organ size and shape. In vaj-1, alterations in the size and shape of floral organs were caused by changes in both cell size and cell number. The vaj-1 mutation also affected the number of floral organs. In vaj-1, a mutation was found in GAMETOPHYTIC FACTOR 1 (GFA1)/CLOTHO (CLO), recently shown to be required for female gametophyte development. The VAJ/GFA1/CLO gene encodes a translational elongation factor-2 (EF-2) family protein, of which the human U5-116 kD and yeast Snu114p counterparts are U5 small nuclear ribonucleoprotein (snRNP)-specific proteins. A transient expression assay using Arabidopsis protoplasts revealed that VAJ protein co-localized with SC35, a serine/arginine-rich (SR) protein involved in pre-mRNA splicing. Our results showed that VAJ/GFA1/CLO has a novel role in the directional control of floral organ growth in Arabidopsis, possibly acting through pre-mRNA splicing.

Physical Interaction of Floral Organs Controls Petal Morphogenesis in Arabidopsis

Smooth petal elongation requires the WS/DGAT family gene FOP1 in Arabidopsis. Flowering plants bear beautiful flowers to attract pollinators. Petals are the most variable organs in flowering plants, with their color, fragrance, and shape. In Arabidopsis (Arabidopsis thaliana), petal primordia arise at a similar time to stamen primordia and elongate at later stages through the narrow space between anthers and sepals. Although many of the genes involved in regulating petal identity and primordia growth are known, the molecular mechanism for the later elongation process remains unknown. We found a mutant, folded petals1 (fop1), in which normal petal development is inhibited during their growth through the narrow space between sepals and anthers, resulting in formation of folded petals at maturation. During elongation, the fop1 petals contact the sepal surface at several sites. The conical-shaped petal epidermal cells are flattened in the fop1 mutant, as if they had been pressed from the top. Surgical or genetic removal of sepals in young buds restores the regular growth of petals, suggesting that narrow space within a bud is the cause of petal folding in the fop1 mutant. FOP1 encodes a member of the bifunctional wax ester synthase/diacylglycerol acyltransferase family, WSD11, which is expressed in elongating petals and localized to the plasma membrane. These results suggest that the FOP1/WSD11 products synthesized in the petal epidermis may act as a lubricant, enabling uninhibited growth of the petals as they extend between the sepals and the anthers.

Axis-dependent Regulation of Lateral Organ Development in Plants

Leaves and floral organs are known as lateral organs formed from meristem at the top of shoots. Distinct from other plant organs, such as stems or roots, the lateral organs are flat with two faces, the adaxial side and the abaxial side. The structural principle of the lateral organs suggests that their development is dependent on three crossing axes, the apical-basal axis, the adaxial-abaxial axis and the lateral axis, although the molecular nature of the axes is not known. Recent studies of Arabidopsis mutant show a couple of examples that putative axes control the expression pattern of genes working in the spatial specification of lateral organs. One of the genes, FILAMENTOUS FLOWER (FIL), a member of the YABBY/FIL gene family encoding a protein with a zinc finger and HMG-related domains, is involved in the specification of the abaxial side of lateral organs. FIL gene expression was restricted at the abaxial side of the lateral organ primordia, suggesting that FIL gene expression is under the control of the putative adaxial-abaxial axis in the lateral organ primordia. Another gene, PRESSED FLOWER (PRS), is a member of the horneobox gene family. Loss of function mutant of PRS lacks two sepals at lateral positions. Two sepals at the adaxial and the abaxial positions are present, but the marginal cell files of the remaining sepals are missing. PRS expression is restricted at the lateral regions of flower primordia and of lateral organs. The expression patterns of PRS strongly suggest that it is controlled by the putative lateral axis formed in the primordia. During development of floral meristems, the axis-dependent expression of FIL and PRS are transiently reduced at stage 2, a stage just before the floral organ primordia appear. Their expression recovers after stage 3, but the expression pattern is different before and after the tentative reduction, suggesting that the center of axes formed in the primordia shifted from the inflorescence meristem to the floral meristem. This shift of the axis center suggests the timing when the floral meristem acquires independence from the inflorescence meristem.