Quantitative phosphoproteomic profiling of fiber differentiation and initiation in a fiberless mutant of cotton

Background

The cotton (Gossypium spp.) fiber cell is an important unicellular model for studying cell differentiation. There is evidence suggesting that phosphorylation is a critical post-translational modification involved in regulation of a wide range of cell activities. Nevertheless, the sites of phosphorylation in G. hirsutum and their regulatory roles in fiber cell initiation are largely unknown. In this study, we employed a mass spectrometry-based phosphoproteomics to conduct a global and site-specific phosphoproteome profiling between ovules of a fuzzless-lintless (fl) Upland cotton (G. hirsutum) mutant and its isogenic parental wild type (WT) at -3 and 0 days post-anthesis (DPA).

Results

A total of 830 phosphopeptides and 1,592 phosphorylation sites from 619 phosphoproteins were identified by iTRAQ (isobaric tags for relative and absolute quantitation). Of these, 76 phosphoproteins and 1,100 phosphorylation sites were identified for the first time after searching the P³DB public database using the BLAST program. Among the detected phosphopeptides, 69 were differentially expressed between the fl mutant and its WT in ovules at -3 and 0 DPA. An analysis using the Motif-X program uncovered 19 phosphorylation motifs, 8 of which were unique to cotton. A further metabolic pathway analysis revealed that the differentially phosphorylated proteins were involved in signal transduction, protein modification, carbohydrate metabolic processes, and cell cycle and cell proliferation.

Conclusions

Our phosphoproteomics-based research provides the first global overview of phosphorylation during cotton fiber initiation, and also offers a helpful dataset for elucidation of signaling networks in fiber development of G. hirsutum.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-466) contains supplementary material, which is available to authorized users.     

Postinfection Development of Rotylenchulus reniformis on Resistant Gossypium barbadense Accessions

The reniform nematode (Rotylenchulus reniformis) causes significant cotton (Gossypium hirsutum) losses in the southeastern United States. The research objective was to describe the effects of two resistant G. barbadense lines (cultivar TX 110 and accession GB 713) on development and fecundity of reniform nematode. Nematode development and fecundity were evaluated on the resistant lines and susceptible G. hirsutum cultivar Deltapine 16 in three repeated growth chamber experiments. Nematode development on roots early and late in the infection cycle was measured at set intervals from 1 to 25 d after inoculation (DAI) and genotypes were compared based on the number of nematodes in four developmental stages (vermiform, swelling, reniform, and gravid). At 15, 20, and 25 DAI, egg production by individual females parasitizing each genotype was measured. Unique reniform nematode developmental patterns were noted on each of the cotton genotypes. During the early stages of infection, infection and development occurred 1 d faster on susceptible cotton than on the resistant genotypes. Later, progression to the reniform and gravid stages of development occurred first on the susceptible genotype, followed by G. barbadense cultivar TX 110, and finally G. barbadense accession GB 713. Egg production by individual nematodes infecting the three genotypes was similar. This study corroborates delayed development previously reported on G. barbadense cultivar TX 110 and is the first report of delayed infection and development associated with G. barbadense accession GB 713. The different developmental patterns in the resistant genotypes suggest that unique or additional loci may confer resistance in these two lines.     

An extensin-rich matrix lines the carinal canals in Equisetum ramosissimum, which may function as water-conducting channels

Background and Aims

The anatomy of Equisetum stems is characterized by the occurrence of vallecular and carinal canals. Previous studies on the carinal canals in several Equisetum species suggest that they convey water from one node to another.

Methods

Cell wall composition and ultrastructure have been studied using immunocytochemistry and electron microscopy, respectively. Serial sectioning and X-ray computed tomography were employed to examine the internode–node–internode transition of Equisetum ramosissimum.

Key Results

The distribution of the LM1 and JIM20 extensin epitopes is restricted to the lining of carinal canals. The monoclonal antibodies JIM5 and LM19 directed against homogalacturonan with a low degree of methyl esterification and the CBM3a probe recognizing crystalline cellulose also bound to this lining. The xyloglucan epitopes recognized by LM15 and CCRC-M1 were only detected in this lining after pectate lyase treatment. The carinal canals, connecting consecutive rings of nodal xylem, are formed by the disruption and dissolution of protoxylem elements during elongation of the internodes. Their inner surface appears smooth compared with that of vallecular canals.

Conclusions

The carinal canals in E. ramosissimum have a distinctive lining containing pectic homogalacturonan, cellulose, xyloglucan and extensin. These canals might function as water-conducting channels which would be especially important during the elongation of the internodes when protoxylem is disrupted and the metaxylem is not yet differentiated. How the molecularly distinct lining relates to the proposed water-conducting function of the carinal canals requires further study. Efforts to elucidate the spatial and temporal distribution of cell wall polymers in a taxonomically broad range of plants will probably provide more insight into the structural–functional relationships of individual cell wall components or of specific configurations of cell wall polymers.     

The distribution of cell wall polymers during antheridium development and spermatogenesis in the Charophycean green alga, Chara corallina

Background and Aims

The production of multicellular gametangia in green plants represents an early evolutionary development that is found today in all land plants and advanced clades of the Charophycean green algae. The processing of cell walls is an integral part of this morphogenesis yet very little is known about cell wall dynamics in early-divergent green plants such as the Charophycean green algae. This study represents a comprehensive analysis of antheridium development and spermatogenesis in the green alga, Chara corallina.

Methods

Microarrays of cell wall components and immunocytochemical methods were employed in order to analyse cell wall macromolecules during antheridium development.

Key Results

Cellulose and pectic homogalacturonan epitopes were detected throughout all cell types of the developing antheridium including the unique cell wall protuberances of the shield cells and the cell walls of sperm cell initials. Arabinogalactan protein epitopes were distributed only in the epidermal shield cell layers and anti-xyloglucan antibody binding was only observed in the capitulum region that initially yields the sperm filaments. During the terminal stage of sperm development, no cell wall polymers recognized by the probes employed were found on the scale-covered sperm cells.

Conclusions

Antheridium development in C. corallina is a rapid event that includes the production of cell walls that contain polymers similar to those found in land plants. While pectic and cellulosic epitopes are ubiquitous in the antheridium, the distribution of arabinogalactan protein and xyloglucan epitopes is restricted to specific zones. Spermatogenesis also includes a major switch in the production of extracellular matrix macromolecules from cell walls to scales, the latter being a primitive extracellular matrix characteristic of green plants.     

A Cotton Annexin Protein AnxGb6 Regulates Fiber Elongation through Its Interaction with Actin 1

Annexins are assumed to be involved in regulating cotton fiber elongation, but direct evidence remains to be presented. Here we cloned six Annexin genes (AnxGb) abundantly expressed in fiber from sea-island cotton (G. barbadense). qRT-PCR results indicated that all six G. barbadense annexin genes were expressed in elongating cotton fibers, while only the expression of AnxGb6 was cotton fiber-specific. Yeast two hybridization and BiFC analysis revealed that AnxGb6 homodimer interacted with a cotton fiber specific actin GbAct1. Ectopic-expressed AnxGb6 in Arabidopsis enhanced its root elongation without increasing the root cell number. Ectopic AnxGb6 expression resulted in more F-actin accumulation in the basal part of the root cell elongation zone. Analysis of AnxGb6 expression in three cotton genotypes with different fiber length confirmed that AnxGb6 expression was correlated to cotton fiber length, especially fiber elongation rate. Our results demonstrated that AnxGb6 was important for fiber elongation by potentially providing a domain for F-actin organization.     

Arabinogalactan protein-rich cell walls,paramural deposits and ergastic globules define the hyaline bodies of rhinanthoid Orobanchaceae haustoria

Background and Aims

Parasitic plants obtain nutrients from their hosts through organs called haustoria. The hyaline body is a specialized parenchymatous tissue occupying the central parts of haustoria in many Orobanchaceae species. The structure and functions of hyaline bodies are poorly understood despite their apparent necessity for the proper functioning of haustoria. Reported here is a cell wall-focused immunohistochemical study of the hyaline bodies of three species from the ecologically important clade of rhinanthoid Orobanchaceae.

Methods

Haustoria collected from laboratory-grown and field-collected plants of Rhinanthus minor, Odontites vernus and Melampyrum pratense attached to various hosts were immunolabelled for cell wall matrix glycans and glycoproteins using specific monoclonal antibodies (mAbs).

Key Results

Hyaline body cell wall architecture differed from that of the surrounding parenchyma in all species investigated. Enrichment in arabinogalactan protein (AGP) epitopes labelled with mAbs LM2, JIM8, JIM13, JIM14 and CCRC-M7 was prominent and coincided with reduced labelling of de-esterified homogalacturonan with mAbs JIM5, LM18 and LM19. Furthermore, paramural bodies, intercellular deposits and globular ergastic bodies composed of pectins, xyloglucans, extensins and AGPs were common. In Rhinanthus they were particularly abundant in pairings with legume hosts. Hyaline body cells were not in direct contact with haustorial xylem, which was surrounded by a single layer of paratracheal parenchyma with thickened cell walls abutting the xylem.

Conclusions

The distinctive anatomy and cell wall architecture indicate hyaline body specialization. Altered proportions of AGPs and pectins may affect the mechanical properties of hyaline body cell walls. This and the association with a transfer-like type of paratracheal parenchyma suggest a role in nutrient translocation. Organelle-rich protoplasts and the presence of exceptionally profuse intra- and intercellular wall materials when attached to a nitrogen-fixing host suggest subsequent processing and transient storage of nutrients. AGPs might therefore be implicated in nutrient transfer and metabolism in haustoria.     

An integrative analysis of four CESA isoforms specific for fiber cellulose production between Gossypium hirsutum and Gossypium barbadense

Cotton fiber is an excellent model system of cellulose biosynthesis; however, it has not been widely studied due to the lack of information about the cellulose synthase (CESA) family of genes in cotton. In this study, we initially identified six full-length CESA genes designated as GhCESA5–GhCESA10. Phylogenetic analysis and gene co-expression profiling revealed that CESA1, CESA2, CESA7, and CESA8 were the major isoforms for secondary cell wall biosynthesis, whereas CESA3, CESA5, CESA6, CESA9, and CESA10 should involve in primary cell wall formation for cotton fiber initiation and elongation. Using integrative analysis of gene expression patterns, CESA protein levels, and cellulose biosynthesis in vivo, we detected that CESA8 could play an enhancing role for rapid and massive cellulose accumulation in Gossypium hirsutum and Gossypium barbadense. We found that CESA2 displayed a major expression in non-fiber tissues and that CESA1, a housekeeping gene like, was predominantly expressed in all tissues. Further, a dynamic alteration was observed in cell wall composition and a significant discrepancy was observed between the cotton species during fiber elongation, suggesting that pectin accumulation and xyloglucan reduction might contribute to cell wall transition. In addition, we discussed that callose synthesis might be regulated in vivo for massive cellulose production during active secondary cell wall biosynthesis in cotton fibers.     

A cytochemical and immunocytochemical analysis of the wall labyrinth apparatus in leaf transfer cells in Elodea canadensis

Background and Aims

Transfer cells are plant cells specialized in apoplast/symplast transport and characterized by a distinctive wall labyrinth apparatus. The molecular architecture and biochemistry of the labyrinth apparatus are poorly known. The leaf lamina in the aquatic angiosperm Elodea canadensis consists of only two cell layers, with the abaxial cells developing as transfer cells. The present study investigated biochemical properties of wall ingrowths and associated plasmalemma in these cells.

Methods

Leaves of Elodea were examined by light and electron microscopy and ATPase activity was localized cytochemically. Immunogold electron microscopy was employed to localize carbohydrate epitopes associated with major cell wall polysaccharides and glycoproteins.

Key Results

The plasmalemma associated with the wall labyrinth is strongly enriched in light-dependent ATPase activity. The wall ingrowths and an underlying wall layer share an LM11 epitope probably associated with glucuronoarabinoxylan and a CCRC-M7 epitope typically associated with rhamnogalacturonan I. No labelling was observed with LM10, an antibody that recognizes low-substituted and unsubstituted xylan, a polysaccharide consistently associated with secondary cell walls. The JIM5 and JIM7 epitopes, associated with homogalacturonan with different degrees of methylation, appear to be absent in the wall labyrinth but present in the rest of cell walls.

Conclusions

The wall labyrinth apparatus of leaf transfer cells in Elodea is a specialized structure with distinctive biochemical properties. The high level of light-dependent ATPase activity in the plasmalemma lining the wall labyrinth is consistent with a formerly suggested role of leaf transfer cells in enhancing inorganic carbon inflow. The wall labyrinth is a part of the primary cell wall. The discovery that the wall ingrowths in Elodea have an antibody-binding pattern divergent, in part, from that of the rest of cell wall suggests that their carbohydrate composition is modulated in relation to transfer cell functioning.     

SNARE VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole and is essential for cell wall organization and root hair growth in arabidopsis

Background and Aims

Root hairs are responsible for water and nutrient uptake from the soil and their growth is responsive to biotic and abiotic changes in their environment. Root hair expansion is a polarized process requiring secretory and endosomal pathways that deliver and recycle plasma membrane and cell wall material to the growing root hair tip. In this paper, the role of VTI13 (AT3G29100), a member of the VTI vesicular soluble NSF attachment receptor (SNARE) gene family in Arabidopsis thaliana, in root hair growth is described.

Methods

Genetic analysis and complementation of the vti13 root hair phenotypes of Arabidopsis thaliana were first used to assess the role of VTI13 in root hair growth. Transgenic lines expressing a green fluorescent protein (GFP)–VTI13 construct were used to characterize the intracellular localization of VTI13 in root hairs using confocal microscopy and immunotransmission electron microscopy.

Key Results

VTI13 was characterized and genetic analysis used to show that its function is required for root hair growth. Expression of a GFP–VTI13 fusion in the vti13 mutant background was shown to complement the vti13 root hair phenotype. GFP–VTI13 localized to both the vacuole membrane and a mobile endosomal compartment. The function of VTI13 was also required for the localization of SYP41 to the trans-Golgi network. Immunohistochemical analysis indicated that cell wall organization is altered in vti13 root hairs and root epidermal cells.

Conclusions

These results show that VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole within root hairs and is essential for the maintenance of cell wall organization and root hair growth in arabidopsis.     

The influence of tetrad shape and intersporal callose wall formation on pollen aperture pattern ontogeny in two eudicot species

Background and Aims

In flowering plants, microsporogenesis is accompanied by various types of cytoplasmic partitioning (cytokinesis). Patterns of male cytokinesis are suspected to play a role in the diversity of aperture patterns found in pollen grains of angiosperms. The relationships between intersporal wall formation, tetrad shape and pollen aperture pattern ontogeny are studied.

Methods

A comparative analysis of meiosis and aperture distribution was performed within tetrads in two triporate eudicot species with contrasting aperture arrangements within their tetrads [Epilobium roseum (Onagraceae) and Paranomus reflexus (Proteaceae)].

Key Results and Conclusions

Intersporal wall formation is a two-step process in both species. Cytokinesis is first achieved by the formation of naked centripetal cell plates. These naked cell plates are then covered by additional thick, localized callose deposits that differ in location between the two species. Apertures are finally formed in areas in which additional callose is deposited on the cell plates. The recorded variation in tetrad shape is correlated with variations in aperture pattern, demonstrating the role of cell partitioning in aperture pattern ontogeny.     

Root cortical aerenchyma inhibits radial nutrient transport in maize (Zea mays)

Background and Aims

Formation of root cortical aerenchyma (RCA) can be induced by nutrient deficiency. In species adapted to aerobic soil conditions, this response is adaptive by reducing root maintenance requirements, thereby permitting greater soil exploration. One trade-off of RCA formation may be reduced radial transport of nutrients due to reduction in living cortical tissue. To test this hypothesis, radial nutrient transport in intact roots of maize (Zea mays) was investigated in two radiolabelling experiments employing genotypes with contrasting RCA.

Methods

In the first experiment, time-course dynamics of phosphate loading into the xylem were measured from excised nodal roots that varied in RCA formation. In the second experiment, uptake of phosphate, calcium and sulphate was measured in seminal roots of intact young plants in which variation in RCA was induced by treatments altering ethylene action or genetic differences.

Key Results

In each of three paired genotype comparisons, the rate of phosphate exudation of high-RCA genotypes was significantly less than that of low-RCA genotypes. In the second experiment, radial nutrient transport of phosphate and calcium was negatively correlated with the extent of RCA for some genotypes.

Conclusions

The results support the hypothesis that RCA can reduce radial transport of some nutrients in some genotypes, which could be an important trade-off of this trait.     

Intraspecific variation in the magnitude and pattern of flooding-induced shoot elongation in Rumex palustris

Background and Aims

Intraspecific variation in flooding tolerance is the basic pre-condition for adaptive flooding tolerance to evolve, and flooding-induced shoot elongation is an important trait that enables plants to survive shallow, prolonged flooding. Here an investigation was conducted to determine to what extent variation in flooding-induced leaf elongation exists among and within populations of the wetland species Rumex palustris, and whether the magnitude of elongation can be linked to habitat characteristics.

Methods

Offspring of eight genotypes collected in each of 12 populations from different sites (ranging from river mudflats with dynamic flooding regimes to areas with stagnant water) were submerged, and petioles, laminas and roots were harvested separately to measure traits related to elongation and plant growth.

Key Results

We found strong elongation of petioles upon submergence, and both among- and within-population variation in this trait, not only in final length, but also in the timing of the elongation response. However, the variation in elongation responses could not be linked to habitat type.

Conclusions

Spatio-temporal variation in the duration and depth of flooding in combination with a presumably weak selection against flooding-induced elongation may have contributed to the maintenance of large genetic variation in flooding-related traits among and within populations.