A cell wall-associated gene network shapes leaf boundary domains.

Boundary domains delimit and organize organ growth throughout plant development almost relentlessly, building plant architecture and morphogenesis. Boundary domains display reduced growth and orchestrate development of adjacent tissues in a non-cell-autonomous manner. How these two functions are achieved remains elusive despite the identification of several boundary-specific genes. Here, we show using morphometrics at the organ and cellular levels that leaf boundary domain development requires SPINDLY (SPY), an O-fucosyltransferase, to act as cell growth repressor. Furthermore, we show that SPY acts redundantly with the CUP-SHAPED COTYLEDON transcription factors (CUC2 and CUC3), which are major determinants of boundaries development. Accordingly, at the molecular level CUC2 and SPY repress a common set of genes involved in cell wall loosening, providing a molecular framework for the growth repression associated with boundary domains. Atomic force microscopy confirmed that young leaf boundary domain cells have stiffer cell walls than marginal outgrowth. This differential cell wall stiffness was reduced in spy mutant plants. Taken together, our data reveal a concealed CUC2 cell wall-associated gene network linking tissue patterning with cell growth and mechanics.

Custom methods to identify conserved genetic modules applied to novel transcriptomic data from Amborella trichopoda.

We have devised a procedure for the inter-species comparison of transcriptomic data and used this procedure to reconstruct the expression dynamics of major genetic modules that were present at least 149 million years ago in the most recent common ancestor of living angiosperms. We began by using laser-assisted microdissection to generate novel transcriptomic data from female flower tissues of Amborella trichopoda, the likely sister to all other living angiosperms. We then employed a gene-expression clustering method, followed by a custom procedure to compare genetic modules on the basis of gene orthology between Amborella and the molecular-genetic model angiosperm Arabidopsis thaliana. Using this protocol, we succeeded in identifying nine major genetic modules that appear to have conserved their expression dynamics from an early stage in angiosperm evolution. The genes of these modules, representing over 5000 orthogroups, include around one third of those known to control female reproductive development in Arabidopsis. Our study constitutes a proof of concept for the comparison of transcriptomic data between widely diverged plant species and represents a first step in the large-scale analysis of gene expression dynamics in a macro-evolutionary context.

Dissecting the pathways coordinating patterning and growth by plant boundary domains.

Boundary domains play important roles during morphogenesis in plants and animals, but how they contribute to patterning and growth coordination in plants is not understood. The CUC genes determine the boundary domains in the aerial part of the plants and, in particular, they have a conserved role in regulating leaf complexity across Angiosperms. Here, we used tooth formation at the Arabidopsis leaf margin controlled by the CUC2 transcription factor to untangle intertwined events during boundary-controlled morphogenesis in plants. Combining conditional restoration of CUC2 function with morphometrics as well as quantification of gene expression and hormone signaling, we first established that tooth morphogenesis involves a patterning phase and a growth phase. These phases can be separated, as patterning requires CUC2 while growth can occur independently of CUC2. Next, we show that CUC2 acts as a trigger to promote growth through the activation of three functional relays. In particular, we show that KLUH acts downstream of CUC2 to modulate auxin response and that expressing KLUH can compensate for deficient CUC2 expression during tooth growth. Together, we reveal a genetic and molecular network that allows coordination of patterning and growth by CUC2-defined boundaries during morphogenesis at the leaf margin.

GDP-L-fucose is required for boundary definition in plants.

The CUP-SHAPED COTYLEDON (CUC) transcription factors control plant boundary formation, thus allowing the emergence of novel growth axes. While the developmental roles of the CUC genes in different organs and across species are well characterized, upstream and downstream events that contribute to their function are still poorly understood. To identify new players in this network, we performed a suppressor screen of CUC2g-m4, a line overexpressing CUC2 that has highly serrated leaves. We identified a mutation that simplifies leaf shape and affects MURUS1 (MUR1), which is responsible for GDP-L-fucose production. Using detailed morphometric analysis, we show that GDP-L-fucose has an essential role in leaf shape acquisition by sustaining differential growth at the leaf margins. Accordingly, reduced CUC2 expression levels are observed in mur1 leaves. Furthermore, genetic analyses reveal a conserved role for GDP-L-fucose in different developmental contexts where it contributes to organ separation in the same pathway as CUC2. Taken together, our results reveal that GDP-L-fucose is necessary for proper establishment of boundary domains in various developmental contexts.

Disruption of LACCASE4 and 17 results in tissue-specific alterations to lignification of Arabidopsis thaliana stems.

Peroxidases have been shown to be involved in the polymerization of lignin precursors, but it remains unclear whether laccases (EC 1.10.3.2) participate in constitutive lignification. We addressed this issue by studying laccase T-DNA insertion mutants in Arabidopsis thaliana. We identified two genes, LAC4 and LAC17, which are strongly expressed in stems. LAC17 was mainly expressed in the interfascicular fibers, whereas LAC4 was expressed in vascular bundles and interfascicular fibers. We produced two double mutants by crossing the LAC17 (lac17) mutant with two LAC4 mutants (lac4-1 and lac4-2). The single and double mutants grew normally in greenhouse conditions. The single mutants had moderately low lignin levels, whereas the stems of lac4-1 lac17 and lac4-2 lac17 mutants had lignin contents that were 20 and 40% lower than those of the control, respectively. These lower lignin levels resulted in higher saccharification yields. Thioacidolysis revealed that disrupting LAC17 principally affected the deposition of G lignin units in the interfascicular fibers and that complementation of lac17 with LAC17 restored a normal lignin profile. This study provides evidence that both LAC4 and LAC17 contribute to the constitutive lignification of Arabidopsis stems and that LAC17 is involved in the deposition of G lignin units in fibers.

Combining laser-assisted microdissection (LAM) and RNA-seq allows to perform a comprehensive transcriptomic analysis of epidermal cells of Arabidopsis embryo.

BACKGROUND: Genome-wide characterization of tissue- or cell-specific gene expression is a recurrent bottleneck in biology. We have developed a sensitive approach based on ultra-low RNA sequencing coupled to laser assisted microdissection for analyzing different tissues of the small Arabidopsis embryo. METHODS AND RESULTS: We first characterized the number of genes detected according to the quantity of tissue yield and total RNA extracted. Our results revealed that as low as 0.02 mm2 of tissue and 50 pg of total RNA can be used without compromising the number of genes detected. The optimised protocol was used to compare the epidermal versus mesophyll cell transcriptomes of cotyledons at the torpedo-shaped stage of embryo development. The approach was validated by the recovery of well-known epidermal genes such AtML1 or AtPDF2 and genes involved in flavonoid and cuticular waxes pathways. Moreover, the interest and sensitivity of this approach were highlighted by the characterization of several transcription factors preferentially expressed in epidermal cells. CONCLUSION: This technical advance unlocks some current limitations of transcriptomic analyses and allows to investigate further and efficiently new biological questions for which only a very small amounts of cells need to be isolated. For instance, it paves the way to increasing the spatial accuracy of regulatory networks in developing small embryo of Arabidopsis or other plant tissues.

Cell expansion-mediated organ growth is affected by mutations in three EXIGUA genes.

Organ growth depends on two distinct, yet integrated, processes: cell proliferation and post-mitotic cell expansion. Although the regulatory networks of plant cell proliferation during organ growth have begun to be unveiled, the mechanisms regulating post-mitotic cell growth remain mostly unknown. Here, we report the characterization of three EXIGUA (EXI) genes that encode different subunits of the cellulose synthase complex specifically required for secondary cell wall formation. Despite this highly specific role of EXI genes, all the cells within the leaf, even those that do not have secondary walls, display small sizes in the exi mutants. In addition, we found a positive correlation between cell size and the DNA ploidy levels in exi mutant leaves, suggesting that both processes share some regulatory components. Our results are consistent with the hypothesis that the collapsed xylem vessels of the exi mutants hamper water transport throughout the plant, which, in turn, limits the turgor pressure levels required for normal post-mitotic cell expansion during leaf growth.