Regulation of shoot epidermal cell differentiation by a pair of homeodomain proteins in Arabidopsis

In higher plants, the outermost cell layer (L1) of the shoot apex gives rise to the epidermis of shoot organs. Our previous study demonstrated that an 8-bp motif named the L1 box functions as a cis-regulatory element for L1-specific gene expression in the shoot system of ARABIDOPSIS: We show here that PROTODERMAL FACTOR2 (PDF2), a member of the HD-GL2 class of homeobox genes, is expressed exclusively in the L1 of shoot meristems and that recombinant PDF2 protein specifically binds to the L1 box in vitro. Although knockout mutants of PDF2 and ATML1, another L1-specific HD-GL2 class gene sharing the highest homology with PDF2, display normal shoot development, the double mutant results in severe defects in shoot epidermal cell differentiation. This suggests that PDF2 and ATML1 are functionally interchangeable and play a critical role in maintaining the identity of L1 cells, possibly by interacting with their L1 box and those of downstream target-gene promoters.     

Allele-specific effects of PDF2 on floral morphology in Arabidopsis thaliana

The class IV Homeodomain-leucine zipper (HD-ZIP IV) gene family includes several genes that are functionally significant in epidermal development. Our recent study revealed that double mutants of the epidermis-expressed HD-ZIP IV members, PROTODERMAL FACTOR2 (PDF2) in combination with some HOMEODOMAIN GLABROUS (HDG, pronounced “hedge”) genes, affect stamen development and specification of petal and stamen identity, possibly in a non cell-autonomous manner. However, the effect of the pdf2 mutations on the floral development was largely different depending on T-DNA insertion locations: pdf2–1 hdg flowers exhibited homeotic conversion of petals and stamens, while pdf2–2 hdg flowers had only a reduced number of stamens. Here, we used 2 additional pdf2 alleles to make double mutants and found that their floral phenotypes were rather similar to those of pdf2–2 hdg. The allele-specific effect caused by pdf2–1, which carries a T-DNA in a steroidogenic acute regulatory protein-related lipid transfer (START) domain-encoding region, suggests the importance of the START domain in proper function of HD-ZIP IV proteins.     

Induction of epidermal cell fate in Arabidopsis shoots

Land plants have evolved a cuticle-bearing epidermis to protect themselves from environmental stress and pathogen attack. Despite its important role, little is known about the molecular mechanisms regulating shoot epidermal cell identity. In a recent study, we found that the Arabidopsis thaliana ATML1 gene is possibly a master regulator of shoot epidermal cell fate. We revealed that ATML1 has the ability to confer shoot epidermis-related traits to non-epidermal cells of the seedlings. These data are consistent with the previous loss-of-function mutant analyses, which implied a positive role of ATML1 in epidermal cell differentiation. Importantly, ectopic epidermal cells induced in ATML1-overexpressing lines provide a novel tool to assess the intrinsic properties of epidermal cells and to study epistatic interactions among genes involved in epidermal/mesophyll differentiation. Using this system, we obtained data revealing that ATML1 negatively influenced mesophyll cell fate. In addition, we provided a working model of how division planes in epidermal cells are determined.     

The Arabidopsis thaliana MERISTEM LAYER 1 promoter specifies epidermal expression in meristems and young primordia

The Arabidopsis thaliana MERISTEM LAYER 1 (ATML1) gene is expressed in the epidermis of developing embryos and shoot meristems. To identify regulatory sequences necessary for epidermis-specific expression, three fusions of overlapping ATML1 genomic sequences to the GUS reporter gene were introduced into Arabidopsis plants. All fusion genes conferred epidermis-specific expression of both GUS mRNA and protein activity but varied in both the timing and relative levels of expression, suggesting partial redundancy of ATML1 regulatory elements. This study defines L1-specific regulatory sequences that are sufficient to direct foreign gene expression in a layer-specific manner.     

Characterization of MORE AXILLARY GROWTH Genes in Populus

Background

Strigolactones are a new class of plant hormones that play a key role in regulating shoot branching. Studies of branching mutants in Arabidopsis, pea, rice and petunia have identified several key genes involved in strigolactone biosynthesis or signaling pathway. In the model plant Arabidopsis, MORE AXILLARY GROWTH1 (MAX1), MAX2, MAX3 and MAX4 are four founding members of strigolactone pathway genes. However, little is known about the strigolactone pathway genes in the woody perennial plants.

Methodology/Principal Finding

Here we report the identification of MAX homologues in the woody model plant Populus trichocarpa. We identified the sequence homologues for each MAX protein in P. trichocarpa. Gene expression analysis revealed that Populus MAX paralogous genes are differentially expressed across various tissues and organs. Furthermore, we showed that Populus MAX genes could complement or partially complement the shoot branching phenotypes of the corresponding Arabidopsis max mutants.

Conclusion/Significance

This study provides genetic evidence that strigolactone pathway genes are likely conserved in the woody perennial plants and lays a foundation for further characterization of strigolactone pathway and its functions in the woody perennial plants.     

AXR2 encodes a member of the Aux/IAA protein family

The dominant gain-of-function axr2-1 mutation of Arabidopsis causes agravitropic root and shoot growth, a short hypocotyl and stem, and auxin-resistant root growth. We have cloned the AXR2 gene using a map-based approach, and find that it is the same as IAA7, a member of the IAA (indole-3-acetic acid) family of auxin-inducible genes. The axr2-1 mutation changes a single amino acid in conserved domain II of AXR2/IAA7. We isolated loss-of-function mutations in AXR2/IAA7 as intragenic suppressors of axr2-1 or in a screen for insertion mutations in IAA genes. A null mutant has a slightly longer hypocotyl than wild-type plants, indicating that AXR2/IAA7 controls development in light-grown seedlings, perhaps in concert with other gene products. Dark-grown axr2-1 mutant plants have short hypocotyls and make leaves, suggesting that activation of AXR2/IAA7 is sufficient to induce morphological responses normally elicited by light. Previously described semidominant mutations in two other Arabidopsis IAA genes cause some of the same phenotypes as axr2-1, but also cause distinct phenotypes. These results illustrate functional differences among members of the Arabidopsis IAA gene family.     

Functional characterization of the Arabidopsis ubiquitin-specific protease gene family reveals specific role and redundancy of individual members in development

Ubiquitin-specific proteases (UBPs) are a highly conserved family of proteins in eukaryotes, and play critical roles in protein de-ubiquitination. Here we report a systematic genetic and expression profiling analysis of the UBP gene family in the Arabidopsis thaliana genome. Mutation analysis of 25 of the 27 member genes representing 13 of the 14 sub-families of the UBP gene family revealed that single-gene mutants of three genes in two sub-families exhibit visible phenotypes. Two of these three genes belonging to the UBP15 sub-family were selected for further characterization. The ubp15 mutants display narrower, serrated and flat rosette leaves, partially due to a defect in cell proliferation, as well as other phenotypes such as early flowering, weak apical dominance and reduced fertility, while the line over-expressing UBP15 shows opposite phenotypes. We demonstrated that UPB15 has UBP activity in vitro , and that this biochemical activity is essential for its in vivo function. A genetic interaction analysis among members of this sub-family revealed that UBP15 and UBP16, but not UBP17, have functional redundancy. Our data thus suggest that distinct UBPs, even within a closely related sub-family, can function in different developmental pathways. Although there are clearly functional redundancies among related sub-family members, those redundancies cannot be inferred simply based on the amino acid identity of the family members.     

CUC1 gene activates the expression of SAM-related genes to induce adventitious shoot formation

CUP-SHAPED COTYLEDON (CUC)1 encodes members of the NAC family. These are functionally redundant genes that are involved in shoot apical meristem (SAM) formation and cotyledon separation during embryogenesis in Arabidopsis. We analyzed transgenic plants overexpressing CUC1 (35S::CUC1). The cotyledons of these transgenic seedlings regularly had two basal lobes, small and round epidermal cells between the sinuses, and adventitious SAMs on the adaxial surface of this region. This suggests that CUC1 promotes adventitious SAM formation by maintaining epidermal cells in an undifferentiated state. In 35S::CUC1 cotyledons, the class I knotted-like homeobox (KNOX) genes, including SHOOT MERISTEMLESS (STM) and BREVIPEDICELLUS (BP), which are involved in SAM formation and/or maintenance, were ectopically expressed before adventitious SAM formation. In stm mutants, ectopic expression of CUC1 could not induce adventitious SAMs, whereas they continued to be observed in bp mutants. These results suggest that STM, but not BP, is necessary for the formation of adventitious SAMs in 35S::CUC1 cotyledons. Furthermore, we examined the relationship between CUC1 and ASYMMETRIC LEAVES (AS)1 and AS2. The as1 and as2 mutations genetically enhance 35S::CUC1 phenotypes even in the absence of STM function. Interestingly, the as1 mutation can partially rescue the mutant vegetative development phenotypes in the cuc1 cuc2 double mutant. Our results suggest that CUC1 positively regulates SAM formation not only through STM but also through an STM-independent pathway that is negatively regulated by AS1 and AS2.     

SGR2, a phospholipase-like protein, and ZIG/SGR4, a SNARE, are involved in the shoot gravitropism of Arabidopsis

In higher plants, the shoot and the root generally show negative and positive gravitropism, respectively. To elucidate the molecular mechanisms involved in gravitropism, we have isolated many shoot gravitropism mutants in Arabidopsis. The sgr2 and zig/sgr4 mutants exhibited abnormal gravitropism in both inflorescence stems and hypocotyls. These genes probably are involved in the early step(s) of the gravitropic response. The sgr2 mutants also had misshapen seed and seedlings, whereas the stem of the zig/sgr4 mutants elongated in a zigzag fashion. The SGR2 gene encodes a novel protein that may be part of a gene family represented by bovine phosphatidic acid-preferring phospholipase A1 containing a putative transmembrane domain. This gene family has been reported only in eukaryotes. The ZIG gene was found to encode AtVTI11, a protein that is homologous with yeast VTI1 and is involved in vesicle transport. Our observations suggest that the two genes may be involved in a vacuolar membrane system that affects shoot gravitropism.     

Rice peptide deformylase PDF1B is crucial for development of chloroplasts

Because protein synthesis begins with N-formylmethionine in plant endosymbiotic organelles, removal of the formyl group by peptide deformylase (PDF) is essential to allowing the excision of the first methionine. Rice contains three copies (OsPDF1A, OsPDF1B and OsPDF1B2) of the PDF genes. Unlike OsPDF1A and OsPDF1B, OsPDF1B2 is apparently non-functional, with several deleterious substitutions and deletions. OsPDF1A is more strongly expressed in the roots, while OsPDF1B is expressed at higher levels in mature leaves. Transient expression of PDF-green fluorescent protein (GFP) fusion proteins in the protoplasts demonstrates that, unlike OsPDF1A, OsPDF1B is localized in both the chloroplasts and the mitochondria. We used T-DNA insertional alleles to elucidate functional roles associated with OsPDF1B. Homozygous plants of pdf1b/pdf1b exhibited the phenotypes of chlorina and growth retardation. Histochemical analysis showed that the length of their mesophyll cells was increased 4- to 5-fold, resulting in a reduction in the total number of cells. Transmission electron microscopy analyses revealed that chloroplasts were severely damaged and mitochondria appeared to be mildly altered in the pdf1b mutants. Expression of genes encoded in the chloroplasts and mitochondria was altered in the mutants. Based on these results, we conclude that OsPDF1B is essential for the development of chloroplast and perhaps mitochondria.