Coordination of differentiation rate and local patterning in compound-leaf development.

The variability in leaf form in nature is immense. Leaf patterning occurs by differential growth, taking place during a limited window of morphogenetic activity at the leaf marginal meristem. While many regulators have been implicated in the designation of the morphogenetic window and in leaf patterning, how these effectors interact to generate a particular form is still not well understood. We investigated the interaction among different effectors of tomato (Solanum lycopersicum) compound-leaf development, using genetic and molecular analyses. Mutations in the tomato auxin response factor SlARF5/SlMP, which normally promotes leaflet formation, suppressed the increased leaf complexity of mutants with extended morphogenetic window. Impaired activity of the NAC/CUC transcription factor GOBLET (GOB), which specifies leaflet boundaries, also reduced leaf complexity in these backgrounds. Analysis of genetic interactions showed that the patterning factors SlMP, GOB and the MYB transcription factor LYRATE (LYR) coordinately regulate leaf patterning by modulating in parallel different aspects of leaflet formation and shaping. This work places an array of developmental regulators in a morphogenetic context. It reveals how organ-level differentiation rate and local growth are coordinated to sculpture an organ. These concepts are applicable to the coordination of pattering and differentiation in other species and developmental processes.

The role of COBRA-LIKE 2 function, as part of the complex network of interacting pathways regulating Arabidopsis seed mucilage polysaccharide matrix organization.

The production of hydrophilic mucilage along the course of seed coat epidermal cell differentiation is a common adaptation in angiosperms. Previous studies have identified COBRA-LIKE 2 (COBL2), a member of the COBRA-LIKE gene family, as a novel component required for crystalline cellulose deposition in seed coat epidermal cells. In recent years, Arabidopsis seed coat epidermal cells (SCEs), also called mucilage secretory cells, have emerged as a powerful model system for the study of plant cell wall components biosynthesis, secretion, assembly and de muro modification. Despite accumulating data, the molecular mechanism of COBL function remains largely unknown. In the current research, we utilized genetic interactions to study the role of COBL2 as part of the protein network required for seed mucilage production. Using correlative phenotyping of structural and biochemical characteristics, unique features of the cobl2 extruded mucilage are revealed, including: 'unraveled' ray morphology, loss of primary cell wall 'pyramidal' organization, reduced Ruthenium red staining intensity of the adherent mucilage layer, and increased levels of the monosaccharides arabinose and galactose. Examination of the cobl2cesa5 double mutant provides insight into the interface between COBL function and cellulose deposition. Additionally, genetic interactions between cobl2 and fei1fei2 as well as between each of these mutants to mucilage-modified 2 (mum2) suggest that COBL2 functions independently of the FEI-SOS pathway. Altogether, the presented data place COBL2 within the complex protein network required for cell wall deposition in the context of seed mucilage and introduce new methodology expending the seed mucilage phenotyping toolbox.

Stomatal cell wall composition: distinctive structural patterns associated with different phylogenetic groups.

Background and Aims: Stomatal morphology and function have remained largely conserved throughout ∼400 million years of plant evolution. However, plant cell wall composition has evolved and changed. Here stomatal cell wall composition was investigated in different vascular plant groups in attempt to understand their possible effect on stomatal function. Methods: A renewed look at stomatal cell walls was attempted utilizing digitalized polar microscopy, confocal microscopy, histology and a numerical finite-elements simulation. The six species of vascular plants chosen for this study cover a broad structural, ecophysiological and evolutionary spectrum: ferns ( Asplenium nidus and Platycerium bifurcatum ) and angiosperms ( Arabidopsis thaliana and Commelina erecta ) with kidney-shaped stomata, and grasses (angiosperms, family Poaceae) with dumbbell-shaped stomata ( Sorghum bicolor and Triticum aestivum ). Key Results: Three distinct patterns of cellulose crystallinity in stomatal cell walls were observed: Type I (kidney-shaped stomata, ferns), Type II (kidney-shaped stomata, angiosperms) and Type III (dumbbell-shaped stomata, grasses). The different stomatal cell wall attributes investigated (cellulose crystallinity, pectins, lignin, phenolics) exhibited taxon-specific patterns, with reciprocal substitution of structural elements in the end-walls of kidney-shaped stomata. According to a numerical bio-mechanical model, the end walls of kidney-shaped stomata develop the highest stresses during opening. Conclusions: The data presented demonstrate for the first time the existence of distinct spatial patterns of varying cellulose crystallinity in guard cell walls. It is also highly intriguing that in angiosperms crystalline cellulose appears to have replaced lignin that occurs in the stomatal end-walls of ferns serving a similar wall strengthening function. Such taxon-specific spatial patterns of cell wall components could imply different biomechanical functions, which in turn could be a consequence of differences in environmental selection along the course of plant evolution.

COBRA-LIKE2, a member of the glycosylphosphatidylinositol-anchored COBRA-LIKE family, plays a role in cellulose deposition in arabidopsis seed coat mucilage secretory cells.

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.

Cellulose synthesis via the FEI2 RLK/SOS5 pathway and cellulose synthase 5 is required for the structure of seed coat mucilage in Arabidopsis.

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.

Abscisic acid plays a key role in the regulation of date palm fruit ripening.

The date palm (Phoenix dactylifera L.) fruit is of major importance for the nutrition of broad populations in the world's desert strip; yet it is sorely understudied. Understanding the mechanism regulating date fruit development and ripening is essential to customise date crop to the climatic change, which elaborates yield losses due to often too early occurring wet season. This study aimed to uncover the mechanism regulating date fruit ripening. To that end, we followed the natural process of date fruit development and the effects of exogenous hormone application on fruit ripening in the elite cultivar 'Medjool'. The results of the current study indicate that the onset of fruit ripening occurre once the seed had reached maximum dry weight. From this point, fruit pericarp endogenous abscisic acid (ABA) levels consistently increased until fruit harvest. The final stage in fruit ripening, the yellow-to-brown transition, was preceded by an arrest of xylem-mediated water transport into the fruit. Exogenous ABA application enhanced fruit ripening when applied just before the green-to-yellow fruit color transition. Repeated ABA applications hastened various fruit ripening processes, resulting in earlier fruit harvest. The data presented supports a pivotal role for ABA in the regulation of date fruit ripening.

Residue Analysis and the Effect of Preharvest Forchlorfenuron (CPPU) Application on On-Tree Quality Maintenance of Ripe Fruit in "Feizixiao" Litchi (Litchi chinensis Sonn.).

Litchi is a highly perishable fruit. Ripe litchi fruit loses quality quickly as they hang on tree, giving a very short hanging life and thus harvest period. This study attempted to explore the roles of cytokinin in regulating fruit ripening and senescence of litchi and examine the possibility of applying cytokinin in "on-tree storage" of the fruit. Exogenous cytokinin, forchlorfenuron (CPPU), was applied at 20 mg L-1 7 weeks after full bloom on litchi (Litchi chinensis cv. Feizixiao) fruit clusters. Color parameters, chlorophylls, anthocyanins, fruit and fruit part weights, total soluble solutes (TSSs), soluble sugars, organic acids, non-anthocyanin flavonoids, ethanol, and also CPPU residue in fruit were traced. CPPU residue was higher but decreased faster in the pericarp than in the aril, where it maintained < 10 µg kg-1. CPPU had no significant effect on fruit weight but tended to increase pericarp weight. The treatment suppressed chlorophyll loss and anthocyanin accumulation in the pericarp, increased non-anthocyanin flavonoids in the aril, but had no significant effects on non-anthocyanin flavonoids in the pericarp and total sugar and organic acids in the aril. As the commercially ripe fruit hanged on tree, TSSs, total sugar, and sucrose decreased with ethanol and acetic acid accumulation in the aril. CPPU significantly suppressed the loss of sucrose and total sugar and the accumulation of ethanol and acetic acid in the aril and inhibited malondialdehyde accumulation in the pericarp of the overripe fruit. Soluble invertase, alcohol dehydrogenase, and pyruvate decarboxylase (PDC) activity and gene expression in the aril were downregulated by CPPU. The results suggest that cytokinin partially suppresses the ripening process in litchi and is effective to slow quality loss in the overripe fruit on tree.

Dual N- and C-terminal processing of citrus chlorophyllase precursor within the plastid membranes leads to the mature enzyme.

Chl, the central player in harvesting light energy for photosynthesis, is enzymatically degraded during natural turnover, leaf senescence, fruit ripening or following biotic/abiotic stress induction. The photodynamic properties of Chl and its metabolites call for tight regulation of the catabolic pathway enzymes to avoid accumulation of intermediate breakdown products. Chlorophyllase, the Chl dephytilation enzyme, was previously demonstrated to be an initiator of Chl breakdown when transcriptionally induced to be expressed during ethylene-induced citrus fruit color break or when heterologously expressed in different plant systems. Citrus chlorophyllase was previously shown to be translated as a precursor protein, which is subsequently post-translationally processed to a mature form. We demonstrate that maturation of citrus chlorophyllase involves dual N- and C-terminal processing which appear to be rate-limiting post-translational events when chlorophyllase expression levels are high. The chlorophyllase precursor and intermediate forms were shown to be of transient nature, while the mature form accumulates over time, suggesting that processing may be involved in post-translational regulation of enzyme in vivo function. This notion is further supported by the finding that neither N- nor C-terminal processed domains are essential for chloroplast targeting of the enzyme, and that both processing events occur within the chloroplast membranes. Studies on the processing of chlorophyllase versions truncated at the N- or C-termini or mutated to abolish C-terminal processing suggest that each of the processing events is independent. Dual N- and C-terminal processing, not involving an organellar targeting signal, has rarely been documented in plants and is unique for a plastid protein.

FASCICLIN-LIKE 18 Is a New Player Regulating Root Elongation in Arabidopsis thaliana.

The plasticity of root development represents a key trait that enables plants to adapt to diverse environmental cues. The pattern of cell wall deposition, alongside other parameters, affects the extent, and direction of root growth. In this study, we report that FASCICLIN-LIKE ARABINOGALACTAN PROTEIN 18 (FLA18) plays a role during root elongation in Arabidopsis thaliana. Using root-specific co-expression analysis, we identified FLA18 to be co-expressed with a sub-set of genes required for root elongation. FLA18 encodes for a putative extra-cellular arabinogalactan protein from the FLA-gene family. Two independent T-DNA insertion lines, named fla18-1 and fla18-2, display short and swollen lateral roots (LRs) when grown on sensitizing condition of high-sucrose containing medium. Unlike fla4/salt overly sensitive 5 (sos5), previously shown to display short and swollen primary root (PR) and LRs under these conditions, the PR of the fla18 mutants is slightly longer compared to the wild-type. Overexpression of the FLA18 CDS complemented the fla18 root phenotype. Genetic interaction between either of the fla18 alleles and sos5 reveals a more severe perturbation of anisotropic growth in both PR and LRs, as compared to the single mutants and the wild-type under restrictive conditions of high sucrose or high-salt containing medium. Additionally, under salt-stress conditions, fla18sos5 had a small, chlorotic shoot phenotype, that was not observed in any of the single mutants or the wild type. As previously shown for sos5, the fla18-1 and fla18-1sos5 root-elongation phenotype is suppressed by abscisic acid (ABA) and display hypersensitivity to the ABA synthesis inhibitor, Fluridon. Last, similar to other cell wall mutants, fla18 root elongation is hypersensitive to the cellulose synthase inhibitor, Isoxaben. Altogether, the presented data assign a new role for FLA18 in the regulation of root elongation. Future studies of the unique vs. redundant roles of FLA proteins during root elongation is anticipated to shed a new light on the regulation of root architecture during plant adaptation to different growth conditions.

Cytokinin treatment modifies litchi fruit pericarp anatomy leading to reduced susceptibility to post-harvest pericarp browning.

Litchi (Litchi chinensis Sonn.) is a subtropical fruit known for its attractive red pericarp color, semi-translucent white aril and unique flavor and aroma. Rapid post-harvest pericarp browning strictly limits litchi fruit marketing. In the current research, we hypothesized that modification of litchi fruit pericarp anatomy by hormone application may reduce fruit susceptibility to post-harvest pericarp browning. In this context, we hypothesized that cytokinin treatment, known to induce cell division, may yield fruit with thicker pericarp and reduced susceptibility for fruit surface micro-crack formation, water loss and post-harvest pericarp browning. Exogenous cytokinin treatment was applied at different stages along the course of litchi fruit development and the effect on fruit pericarp anatomy, fruit maturation and postharvest pericarp browning was investigated. Interestingly, cytokinin treatment, applied 4 weeks after full female bloom (WFB), during the phase of pericarp cell division, led to mature fruit with thicker pericarp, reduced rate of post-harvest water loss and reduced susceptibility to post-harvest pericarp browning, as compared to non-treated control fruit. Histological sections ascribe the difference in pericarp anatomy to increased cell proliferation in the parenchymatic tissue and the highly-lignified brachysclereid cell layer. In contrast, exogenous cytokinin treatment applied 7 WFB, following the phase of pericarp cell division, significantly increased epidermal-cell proliferation but had no significant effect on overall fruit pericarp thickness and only minor affect on post-harvest water loss or pericarp browning. Interestingly, the late cytokinin treatment also significantly postponed fruit maturation-associated anthocyanin accumulation and chlorophyll degradation, as previously reported, but had no effect on other parameters of fruit maturation, like total soluble sugars and total titratable acids typically modified during aril maturation. In conclusion, exogenous cytokinin treatment at different stages in fruit development differentially modifies litchi fruit pericarp anatomy by induction of cell-type specific cell proliferation. Early cytokinin treatment during the phase of pericarp cell division may prolong litchi fruit storage by reducing fruit susceptibility to post-harvest water loss and pericarp browning.

Permanently open stomata of aquatic angiosperms display modified cellulose crystallinity patterns.

Most floating aquatic plants have stomata on their upper leaf surfaces, and usually their stomata are permanently open. We previously identified 3 distinct crystallinity patterns in stomatal cell walls, with angiosperm kidney-shaped stomata having the highest crystallinity in the polar end walls as well as the adjacent polar regions of the guard cells. A numerical bio-mechanical model suggested that the high crystallinity areas are localized to regions where the highest stress is imposed. Here, stomatal cell wall crystallinity was examined in 4 floating plants from 2 different taxa: basal angiosperms from the ANITA grade and monocots. It appears that the non-functional stomata of floating plants display reduced crystallinity in the polar regions as compared with high crystallinity of the ventral (inner) walls. Thus their guard cells are both less flexible and less stress resistant. Our findings suggest that the pattern of cellulose crystallinity in stomata of floating plants from different families was altered as a consequence of similar evolutionary pressures.

The FEI2-SOS5 pathway and CELLULOSE SYNTHASE 5 are required for cellulose biosynthesis in the Arabidopsis seed coat and affect pectin mucilage structure.

A common adaptation in angiosperms is the deposition of hydrophilic mucilage into the apoplast of seed coat epidermal cells during the course of their differentiation. Upon imbibition, seed mucilage, composed mainly of carbohydrates (i.e. pectins, hemicelluloses and glycans) expands rapidly, encapsulating the seed and aiding in seed dispersal and germination. The FEI1/FEI2 receptor-like kinases and the SOS5 extracellular GPI-anchored protein were previously shown to act on a pathway regulating cellulose biosynthesis during Arabidopsis root elongation. In the highlighted study, we demonstrated that FEI2 and SOS5 regulate the production of the cellulosic rays deposited across the inner adherent-layer of seed mucilage. Mutations in either fei2 or sos5 disrupted the formation of rays, which was associated with an increase in the soluble, outer layer of pectin mucilage and accompanied by a reduction in the inner adherent-layer. Mutations in CELLULOSE SYNTHASE 5 also led to reduced rays and mal-partitioning of the pectic component of seed mucilage, further establishing a structural role for cellulose in seed mucilage. Here, we show that FEI2 expressed from a CaMV 35S promoter complemented both root and seed mucilage defects of the fei1 fei2 double mutant. In contrast, expression of FEI1 from a 35S promoter complemented the root, but not the seed phenotype of the fei1 fei2 double mutant, suggesting that unlike in the root, FEI2 plays a unique and non-redundant role in the regulation of cellulose synthesis in seed mucilage. Altogether, these data suggest a novel role for cellulose in anchoring the pectic component of seed mucilage to the seed surface and indicate that the FEI2 protein has a function distinct from that of FEI1, despite the high sequence similarity of these RLKs.

The NAC-domain transcription factor GOBLET specifies leaflet boundaries in compound tomato leaves.

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) loss-of-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. Leaf-specific 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.

Citrus chlorophyllase dynamics at ethylene-induced fruit color-break: a study of chlorophyllase expression, posttranslational processing kinetics, and in situ intracellular localization.

Fruit color-break is the visual manifestation of the developmentally regulated transition of chloroplasts to chromoplasts during fruit ripening and often involves biosynthesis of copious amounts of carotenoids concomitant with massive breakdown of chlorophyll. Regulation of chlorophyll breakdown at different physiological and developmental stages of the plant life cycle, particularly at fruit color-break, is still not well understood. Here, we present the dynamics of native chlorophyllase (Chlase) and chlorophyll breakdown in lemon (Citrus limon) fruit during ethylene-induced color-break. We show, using in situ immunofluorescence on ethylene-treated fruit peel (flavedo) tissue, that citrus Chlase is located in the plastid, in contrast to recent reports suggesting cytoplasmic localization of Arabidopsis (Arabidopsis thaliana) Chlases. At the intra-organellar level, Chlase signal was found to overlap mostly with chlorophyll fluorescence, suggesting association of most of the Chlase protein with the photosynthetic membranes. Confocal microscopy analysis showed that the kinetics of chlorophyll breakdown was not uniform in the flavedo cells. Chlorophyll quantity at the cellular level was negatively correlated with plastid Chlase accumulation; plastids with reduced chlorophyll content were found by in situ immunofluorescence to contain significant levels of Chlase, while plastids containing still-intact chlorophyll lacked any Chlase signal. Immunoblot and protein-mass spectrometry analyses were used to demonstrate that citrus Chlase initially accumulates as an approximately 35-kD precursor, which is subsequently N-terminally processed to approximately 33-kD mature forms by cleavage at either of three consecutive amino acid positions. Chlase plastid localization, expression kinetics, and the negative correlation with chlorophyll levels support the central role of the enzyme in chlorophyll breakdown during citrus fruit color-break.

Chlorophyllase is a rate-limiting enzyme in chlorophyll catabolism and is posttranslationally regulated.

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 (ChlaseDeltaN), which corresponds to the mature protein, led to extensive chlorophyll breakdown in both tobacco protoplasts and squash leaves. ChlaseDeltaN-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. Full-length 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.