Leaves are formed at the flanks of the shoot apical meristem (SAM) and develop into a variety of forms. In tomato, prolonged leaf patterning enables the elaboration of compound leaves by reiterative initiation of leaflets with lobed margins. In goblet (gob) lossof-function mutants, primary leaflets are often fused, secondary leaflets and marginal serrations are absent, and SAMs often terminate precociously. We show that GOB encodes a NAC-domain transcription factor expressed in narrow stripes at the leaf margins, flanking the distal side of future leaflet primordia, and at the boundaries between the SAM and leaf primordia. Leafspecific overexpression of the microRNA miR164, a negative regulator of GOB-like genes, also leads to loss of secondary-leaflet initiation and to smooth leaflet margins. Plants carrying a dominant gob allele with an intact ORF but disrupted miR164 binding site produce more cotyledons and floral organs, have split SAMs and, surprisingly, simpler leaves. Overexpression of a form of GOB with an altered miR164 binding site in leaf primordia leads to delayed leaflet maturation, frequent, improperly timed and spaced initiation events, and a simple mature leaflet form owing to secondary-leaflet fusion. miR164 also affects leaflet separation in Cardamine hirsuta, a Brassicaceae species with complex leaves. Genetic and molecular analyses suggest that GOB expression is intact in the simplified leaves of entire tomato mutants, which have a defect in a putative repressor of auxin responses. Our results show that GOB marks leaflet boundaries and that its accurate spatial, temporal and quantitative activity affects leaf elaboration in a context-dependent manner.

The seeds of Arabidopsis thaliana and many other plants are surrounded by a pectinaceous mucilage that aids in seed hydration and germination. Mucilage is synthesized during seed development within maternally derived seed coat mucilage secretory cells (MSCs), and is released to surround the seed upon imbibition. The FEI1/FEI2 receptor-like kinases and the SOS5 extracellular GPI-anchored protein were shown previously to act on a pathway that regulates the synthesis of cellulose in Arabidopsis roots. Here, we demonstrate that both FEI2 and SOS5 also play a role in the synthesis of seed mucilage. Disruption of FEI2 or SOS5 leads to a reduction in the rays of cellulose observed across the seed mucilage inner layer, which alters the structure of the mucilage in response to hydration. Mutations in CESA5, which disrupts an isoform of cellulose synthase involved in primary cell wall synthesis, result in a similar seed mucilage phenotype. The data indicate that CESA5-derived cellulose plays an important role in the synthesis and structure of seed coat mucilage and that the FEI2/SOS5 pathway plays a role in the regulation of cellulose synthesis in MSCs. Moreover, these results establish a novel structural role for cellulose in anchoring the pectic component of seed coat mucilage to the seed surface.

Chlorophyll is a central player in harvesting light energy for photosynthesis, yet the rate-limiting steps of chlorophyll catabolism and the regulation of the catabolic enzymes remain unresolved. To study the role and regulation of chlorophyllase (Chlase), the first enzyme of the chlorophyll catabolic pathway, we expressed precursor and mature versions of citrus (Citrus sinensis) Chlase in two heterologous plant systems: (1) squash (Cucurbita pepo) plants using a viral vector expression system; and (2) transiently transformed tobacco (Nicotiana tabacum) protoplasts. Expression of full-length citrus Chlase resulted in limited chlorophyll breakdown in protoplasts and no visible leaf phenotype in whole plants, whereas expression of a Chlase version lacking the N-terminal 21 amino acids (ChlaseDN), which corresponds to the mature protein, led to extensive chlorophyll breakdown in both tobacco protoplasts and squash leaves. ChlaseDN-expressing squash leaves displayed a dramatic chlorotic phenotype in plants grown under low-intensity light, whereas under natural light a lesion-mimic phenotype occurred, which was demonstrated to follow the accumulation of chlorophyllide, a photodynamic chlorophyll breakdown product. Fulllength and mature citrus Chlase versions were localized to the chloroplast membrane fraction in expressing tobacco protoplasts, where processing of the N-terminal 21 amino acids appears to occur. Results obtained in both plant systems suggest that Chlase functions as a rate-limiting enzyme in chlorophyll catabolism controlled via posttranslational regulation.

Differentiation of the maternally derived seed coat epidermal cells into mucilage secretory cells is a common adaptation in angiosperms. Recent studies identified cellulose as an important component of seed mucilage in various species. Cellulose is deposited as a set of rays that radiate from the seed upon mucilage extrusion, serving to anchor the pectic component of seed mucilage to the seed surface. Using transcriptome data encompassing the course of seed development, we identified COBRA-LIKE2 (COBL2), a member of the glycosylphosphatidylinositol-anchored COBRA-LIKE gene family in Arabidopsis (Arabidopsis thaliana), as coexpressed with other genes involved in cellulose deposition in mucilage secretory cells. Disruption of the COBL2 gene results in substantial reduction in the rays of cellulose present in seed mucilage, along with an increased solubility of the pectic component of the mucilage. Light birefringence demonstrates a substantial decrease in crystalline cellulose deposition into the cellulosic rays of the cobl2 mutants. Moreover, crystalline cellulose deposition into the radial cell walls and the columella appears substantially compromised, as demonstrated by scanning electron microscopy and in situ quantification of light birefringence. Overall, the cobl2 mutants display about 40% reduction in whole-seed crystalline cellulose content compared with the wild type. These data establish that COBL2 plays a role in the deposition of crystalline cellulose into various secondary cell wall structures during seed coat epidermal cell differentiation.