Secondary Cell Wall Development Research Articles

Endo-β-1,4-glucanases impact plant cell wall development by influencing cellulose crystallization.

Cell walls are vital to the normal growth and development of plants as they protect the protoplast and provide rigidity to the stem. Here, two poplar and Arabidopsis orthologous endoglucanases, which have been proposed to play a role in secondary cell wall development, were examined. The class B endoglucanases, PtGH9B5 and AtGH9B5, are secreted enzymes that have a predicted glycosylphosphatidylinositol anchor, while the class C endoglucanases, PtGH9C2 and AtGH9C2, are also predicted to be secreted but instead contain a carbohydrate-binding module. The poplar endoglucanases were expressed in Arabidopsis using both a 35S promoter and the Arabidopsis secondary cell wall-specific CesA8 promoter. Additionally, Arabidopsis t-DNA insertion lines and an RNAi construct was created to downregulate AtGH9C2 in Arabidopsis. All of the plant lines were examined for changes in cell morphology and patterning, growth and development, cell wall crystallinity, microfibril angle, and proportion of cell wall carbohydrates. Misregulation of PtGH9B5/AtGH9B5 resulted in changes in xylose content, while misregulation of PtGH9C2/AtGH9C2 resulted in changes in crystallinity, which was inversely correlated with changes in plant height and rosette diameter. Together, these results suggest that these endoglucanases affect secondary cell wall development by contributing to the cell wall crystallization process.

FT-IR Examination of the Development of Secondary Cell Wall in Cotton Fibers

The secondary cell wall development of cotton fibers harvested at 18, 20, 24, 28, 32, 36 and 40 days after flowering was examined using attenuated total reflection Fourier transform-infrared (ATR FT-IR) spectroscopy. Spectra of deuterated cotton fibers did not demonstrate significant changes in their O–H stretching band shapes or positions during development. Only a progressive increase in O–H band intensity was observed. Results indicate that the highly crystalline cellulose component produced during secondary cell wall formation maintains the hydrogen bonding network observed for the primary cell wall. Other general changes were observed for the regular ATR spectra. A progressive intensity increase for bands assigned to cellulose Iβ was observed during fiber development, including a marked intensity increase for vibrations at 1002 and 985 cm−1. In contrast, C–O vibrational bands from dominant conformations observed at 1104, 1052, 1028 cm−1 undergo a modest intensity increase during secondary cell wall development.

The NAC transcription factor OsSWN1 regulates secondary cell wall development in Oryza sativa

Rice, as a major crop in the world, produces huge agronomic biomass residues besides food, which consist of cellulose, hemicelluloses and lignin. Many master regulators of secondary wall synthesis were identified in the model plant Arabidopsis. In this study, we investigated the function of a NAC (NAM, ATAF, and CUC2) transcription factor related to secondary cell wall biosynthesis, which is highly expressed in rice sclerenchyma tissue and is named OsSWN1. Our results showed that engineering of OsSWN1 could exhibit multiple features regulated to agronomic traits and bioenergy research. Over-expression of OsSWN1 caused an erect-leaf and enclosed-flower phenotype. Secondary cell wall-related genes were actively expressed in transgenic plants with obvious ectopic lignin deposition in the leaf collar, while increased lignin content and decreased the sugar yield correspondingly. In addition, down-regulation of OsSWN1 expression levels decreased lignin content and increased the sugar yield in transgenic plants. Bioinformatics analysis revealed that OsSWN1-like genes are highly conserved in switchgrass and sorghum, suggesting a possibility of manipulating the expression level of the OsSWN1 orthologs in the bioenergy crops for biofuel production.

Effects of elevated temperature on sucrose metabolism and cellulose synthesis in cotton fibre during secondary cell wall development.

Global warming has the potential to increase air temperatures by 1.8 to 4.0°C by the end of the 21st century. In order to reveal the effects of increased temperatures on the sucrose metabolism and cellulose synthesis in cotton fibre during its flowering and boll formation stage, field experiments with elevated temperature regimes (32.6/28.6°C, mean daytime/night-time temperature during flowering and boll formation stage during 2010-12, the same below) and ambient temperature regimes (30.1/25.8°C) were conducted. Activities of sucrose synthase and acid/alkaline invertase decreased under elevated temperature in fibre, but activities of sucrose phosphate synthase were increased. Callose content increased, but sucrose content decreased within the cotton fibre under elevated temperature. The disparity of callose content and sucrose content between the two temperature regimes decreased with the number of days post anthesis, indicating that the effects of elevated temperature on both sucrose content and cellulose content were diminished as the boll matured. Due to the dynamics of the carbohydrate content and associated enzyme activities, we hypothesise that the restrained sucrose metabolism and cellulose biosynthesis under elevated temperatures were mainly attributed to the changed activities of sucrose synthase and invertase. Furthermore, 32.6/28.6°C had a negative effect on the cellulose synthesis compared with 30.1/25.8°C.

BEL1-LIKE HOMEODOMAIN6 and KNOTTED ARABIDOPSIS THALIANA7 interact and regulate secondary cell wall formation via repression of REVOLUTA.

The TALE homeodomain transcription factor KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) is part of a regulatory network governing the commitment to secondary cell wall biosynthesis of Arabidopsis thaliana, where it contributes to negative regulation of this process. Here, we report that BLH6, a BELL1-LIKE HOMEODOMAIN protein, specifically interacts with KNAT7, and this interaction influences secondary cell wall development. BLH6 is a transcriptional repressor, and BLH6-KNAT7 physical interaction enhances KNAT7 and BLH6 repression activities. The overlapping expression patterns of BLH6 and KNAT7 and phenotypes of blh6, knat7, and blh6 knat7 loss-of-function mutants are consistent with the existence of a BLH6-KNAT7 heterodimer that represses commitment to secondary cell wall biosynthesis in interfascicular fibers. BLH6 and KNAT7 overexpression results in thinner interfascicular fiber secondary cell walls, phenotypes that are dependent on the interacting partner. A major impact of the loss of BLH6 and KNAT7 function is enhanced expression of the homeodomain-leucine zipper transcription factor REVOLUTA/INTERFASCICULAR FIBERLESS1 (REV/IFL1). BLH6 and KNAT7 bind to the REV promoter and repress REV expression, while blh6 and knat7 interfascicular fiber secondary cell wall phenotypes are suppressed in blh6 rev and knat7 rev double mutants, suggesting that BLH6/KNAT7 signaling acts through REV as a direct target.

Brassinosteroid signaling affects secondary cell wall deposition in cotton fibers

Differentiation of cotton fibers involves sequential cell elongation and secondary cell wall deposition as they develop from fiber initials to highly elongated and thickened trichomes on the seed integument. Phytohormones appear to play important regulatory roles in the development of these economically important cells. Previous pharmacological experiments indicate that brassinosteroid (BR), along with auxin and gibberellins, promote cotton fiber elongation. To further evaluate the role of the BR signaling pathway in cotton fiber development, transgenic cotton plants that express transgenes designed to either over-express or suppress the expression of the BR receptor BRI1 were created. Analysis of the cotton fibers from these plants indicated that alteration of BRI1 expression had little effect on fiber length. However, over-expression of BRI1 led to increased fiber cellulose deposition while suppression of BRI1 expression strongly inhibited secondary cell wall development, resulting in fibers with reduced maturity. These alterations in fiber cell wall development corresponded with changes in cellulose synthase gene expression, indicating that secondary wall deposition is affected by BR-dependent modulation of cellulose synthase gene expression. These results indicate that BR signaling promotes the maturation of cotton fibers through the deposition of cellulose into the secondary cell wall.

Molecular Cloning of Hypervariable Regions (HVRII) from Cellulose Synthase (CesA) Gene in Neolamarckia Cadamba

Neolamarckia cadamba or locally known as Kelampayan, is one of the fast growing plantation tree species that holds great prospect as a renewable bioresources for plywood, pulp and paper, and biofuel industries. Sufficient information on cellulose synthase (CesA) gene, especially the hypervariable region II (HVRII) component involved in wood formation of Kelampayan is imperative for future applications. This region is thought to play a role in interaction with other unique cell-type-specific proteins involved in the biosynthesis of cellulose. The aim of this study was to identify and clone the HVRII regions of cellulose synthase gene from the developing xylem tissues of Kelampayan. The cDNA of cellulose synthase HVRII regions was amplified using reverse transcription-PCR (RT-PCR) approach using degenerate primers. Three clones, namely NcCesA1HVRII (520bp), NcCesA2HVRII (580bp) and NcCesA3HVRII (620bp) were successfully sequenced and characterized. NcCesA1HVRII and NcCesA3HVRII were clustered into two distinct clades implicated with secondary cell wall development whereas NcCesA2HVRII has renamed to NcCslD1HVRII due its high similarity with various plants’ CslD-HVRII. This study provides an easier and faster access to NcCesAHVRII sequences to further understand the role of NcCesA/NcCslD protein for future applications such as selecting trees with optimal cellulose content required for specific industries.

Xylem sap in cotton contains proteins that contribute to environmental stress response and cell wall development.

The xylem sap of a plant is primarily responsible for transporting molecules from the underground root system to the aboveground parts of the plant body. In order to understand the role that roots play in cotton growth and development, the components present in xylem sap must be elucidated. In this study, we used a shotgun HPLC-ESI-MS/MS proteomics approach to identify 455 peptides from the xylem sap of field-grown cotton plants at peak blooming stage. Of these peptides, 384 (84.4%) were found to be secreted proteins and 320 (70.3%) had special molecular functions. Based on Gene Ontology (GO) analysis, 348 peptides were annotated in terms of molecular function, biological process, and cellular localization, with 46.9 and 45.1% being related to catalytic activity and binding activity, respectively. Many xylem sap-containing proteins were predicted to be involved in different phases of xylem differentiation including cell wall metabolism, secondary cell wall development and patterning, and programmed cell death. The identification of starch and sucrose hydrolyzing enzymes implicated the interaction between roots and aboveground parts on the aspect of carbohydrate metabolism. Many of the proteins identified in this study are involved in defense mechanisms including pathogen-related proteins, such as peroxidases, chitinases, and germin-like proteins, proteases involved in disease resistance, and phytoalexin phenylpropanoid synthesis-related proteins. The majority of identified signaling proteins were fasciclin-like arabinogalactan proteins and kinases. The results of this study provide useful insight into the communication mechanisms between cotton roots and the rest of the cotton plant.

Emerging roles of small GTPases in secondary cell wall development.

Regulation of plant cell wall deposition and patterning is essential for the normal growth and development of plants. Small GTPases play pivotal roles in the modulation of primary cell wall formation by controlling cytoskeletal organization and membrane trafficking. However, the functions of small GTPases in secondary cell wall development are poorly understood. Recent studies on xylem cells revealed that the Rho of plants (ROP) group of small GTPases critically participates in the spatial patterning of secondary cell walls. In differentiating xylem cells, a specific GTPase-activating protein (GAP)/guanine nucleotide exchange factor (GEF) pair facilitates local activation of ROP11 to establish de novo plasma membrane domains. The activated ROP11 then recruits a microtubule-associated protein, MIDD1, to mediate the mutual inhibition between cortical microtubules and active ROP. Furthermore, recent works suggest that certain small GTPases, including ROP and Rab GTPases, regulate membrane trafficking to establish secondary cell wall deposition and patterning. Accordingly, this mini-review assesses and summarizes the current literature regarding the emerging functions of small GTPases in the development of secondary cell walls.

Laccases direct lignification in the discrete secondary cell wall domains of protoxylem.

Plants precisely control lignin deposition in spiral or annular secondary cell wall domains during protoxylem tracheary element (TE) development. Because protoxylem TEs function to transport water within rapidly elongating tissues, it is important that lignin deposition is restricted to the secondary cell walls in order to preserve the plasticity of adjacent primary wall domains. The Arabidopsis (Arabidopsis thaliana) inducible VASCULAR NAC DOMAIN7 (VND7) protoxylem TE differentiation system permits the use of mutant backgrounds, fluorescent protein tagging, and high-resolution live-cell imaging of xylem cells during secondary cell wall development. Enzymes synthesizing monolignols, as well as putative monolignol transporters, showed a uniform distribution during protoxylem TE differentiation. By contrast, the oxidative enzymes LACCASE4 (LAC4) and LAC17 were spatially localized to secondary cell walls throughout protoxylem TE differentiation. These data support the hypothesis that precise delivery of oxidative enzymes determines the pattern of cell wall lignification. This view was supported by lac4lac17 mutant analysis demonstrating that laccases are necessary for protoxylem TE lignification. Overexpression studies showed that laccases are sufficient to catalyze ectopic lignin polymerization in primary cell walls when exogenous monolignols are supplied. Our data support a model of protoxylem TE lignification in which monolignols are highly mobile once exported to the cell wall, and in which precise targeting of laccases to secondary cell wall domains directs lignin deposition.

Secondary cell wall composition and candidate gene expression in developing willow (Salix purpurea) stems

The properties of the secondary cell wall (SCW) in willow largely determine the suitability of willow biomass feedstock for potential bioenergy and biofuel applications. SCW development has been little studied in willow and it is not known how willow compares with model species, particularly the closely related genus Populus. To address this and relate SCW synthesis to candidate genes in willow, a tractable bud culture-derived system was developed in Salix purpurea, and cell wall composition and RNA-Seq transcriptome were followed in stems during early development. A large increase in SCW deposition in the period 0–2 weeks after transfer to soil was characterised by a big increase in xylan content, but no change in the frequency of substitution of xylan with glucuronic acid, and increased abundance of putative transcripts for synthesis of SCW cellulose, xylan and lignin. Histochemical staining and immunolabeling revealed that increased deposition of lignin and xylan was associated with xylem, xylem fibre cells and phloem fibre cells. Transcripts orthologous to those encoding xylan synthase components IRX9 and IRX10 and xylan glucuronyl transferase GUX1 in Arabidopsis were co-expressed, and showed the same spatial pattern of expression revealed by in situ hybridisation at four developmental stages, with abundant expression in proto-xylem, xylem fibre and ray parenchyma cells and some expression in phloem fibre cells. The results show a close similarity with SCW development in Populus species, but also give novel information on the relationship between spatial and temporal variation in xylan-related transcripts and xylan composition.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-014-2034-1) contains supplementary material, which is available to authorized users.

Aberrant phenotype and transcriptome expression during fiber cell wall thickening caused by the mutation of the Im gene in immature fiber (im) mutant in Gossypium hirsutum L.

BackgroundThe immature fiber (im) mutant of Gossypium hirsutum L. is a special cotton fiber mutant with non-fluffy fibers. It has low dry weight and fineness of fibers due to developmental defects in fiber secondary cell wall (SCW).ResultsWe compared the cellulose content in fibers, thickness of fiber cell wall and fiber transcriptional profiling during SCW development in im mutant and its near-isogenic wild-type line (NIL) TM-1. The im mutant had lower cellulose content and thinner cell walls than TM-1 at same fiber developmental stage. During 25 ~ 35 day post-anthesis (DPA), sucrose content, an important carbon source for cellulose synthesis, was also significantly lower in im mutant than in TM-1. Comparative analysis of fiber transcriptional profiling from 13 ~ 25 DPA indicated that the largest transcriptional variations between the two lines occurred at the onset of SCW development. TM-1 began SCW biosynthesis approximately at 16 DPA, whereas the same fiber developmental program in im mutant was delayed until 19 DPA, suggesting an asynchronous fiber developmental program between TM-1 and im mutant. Functional classification and enrichment analysis of differentially expressed genes (DEGs) between the two NILs indicated that genes associated with biological processes related to cellulose synthesis, secondary cell wall biogenesis, cell wall thickening and sucrose metabolism, respectively, were significantly up-regulated in TM-1. Twelve genes related to carbohydrate metabolism were validated by quantitative reverse transcription PCR (qRT-PCR) and confirmed a temporal difference at the earlier transition and SCW biosynthesis stages of fiber development between TM-1 and im mutant.ConclusionsWe propose that Im is an important regulatory gene influencing temporal differences in expression of genes related to fiber SCW biosynthesis. This study lays a foundation for cloning the Im gene, elucidating molecular mechanism of fiber SCW development and further genetic manipulation for the improvement of fiber fineness and maturity.

LuFLA1PRO and LuBGAL1PRO promote gene expression in the phloem fibres of flax (Linum usitatissimum)

Cell type-specific promoters were identified that drive gene expression in an industrially important product. To identify flax (Linum usitatissimum) gene promoters, we analyzed the genomic regions upstream of a fasciclin-like arabinogalactan protein (LuFLA1) and a beta-galactosidase (LuBGAL1). Both of these genes encode transcripts that have been found to be highly enriched in tissues bearing phloem fibres. Using a beta-glucuronidase (GUS) reporter construct, we found that a 908-bp genomic sequence upstream of LuFLA1 (LuFLA1PRO) directed GUS expression with high specificity to phloem fibres undergoing secondary cell wall development. The DNA sequence upstream of LuBGAL1 (LuBGAL1PRO) likewise produced GUS staining in phloem fibres with developing secondary walls, as well as in tissues of developing flowers and seed bolls. These data provide further evidence of a specific role for LuFLA1 in phloem fibre development, and demonstrate the utility of LuFLA1PRO and LuBGAL1PRO as tools for biotechnology and further investigations of phloem fibre development.

Immunolocalization of hemicelluloses in Arabidopsis thaliana stem. Part I: temporal and spatial distribution of xylans

We investigated the microdistribution of xylans in different cell types of Arabidopsis stem using immunolocalization methods with LM10 and LM11 antibodies. Xylan labeling in xylary fibers (fibers) was initially detected at the cell corner of the S(1) layer and increased gradually during fiber maturation, showing correlation between xylan labeling and general secondary cell wall formation processes in fibers. Metaxylem vessels (vessels) showed earlier development of secondary cell walls than fibers, but revealed almost identical labeling patterns to fibers during maturation. No difference in labeling patterns and intensity was detected in the cell wall of fibers, vessels and protoxylem vessels (proto-vessels) between LM10 and LM11, indicating that vascular bundle cells may be chemically composed of a highly homogeneous xylan type. Interestingly, interfascicular fibers (If-fibers) showed different labeling patterns between the two antibodies and also between different developmental stages. LM10 showed no labeling in primary cell walls and intercellular layers of If-fibers at the S(1) formation stage, but some labeling was detected in middle lamella cell corner regions at the S(2) formation stage. In contrast, LM11 revealed uniform labeling across the If-fiber cell wall during all developmental stages. These results suggest that If-fibers have different xylan deposition processes and patterns from vascular bundle cells. The presence of xylan was also confirmed in parenchyma cells following pectinase treatment. Together our results indicate that there are temporal and spatial differences in xylan labeling between cell types in Arabidopsis stem. Differences in xylan labeling between Arabidopsis stem and poplar are also discussed.

SND2, a NAC transcription factor gene, regulates genes involved in secondary cell wall development in Arabidopsis fibres and increases fibre cell area in Eucalyptus

BackgroundNAC domain transcription factors initiate secondary cell wall biosynthesis in Arabidopsis fibres and vessels by activating numerous transcriptional regulators and biosynthetic genes. NAC family member SND2 is an indirect target of a principal regulator of fibre secondary cell wall formation, SND1. A previous study showed that overexpression of SND2 produced a fibre cell-specific increase in secondary cell wall thickness in Arabidopsis stems, and that the protein was able to transactivate the cellulose synthase8 (CesA8) promoter. However, the full repertoire of genes regulated by SND2 is unknown, and the effect of its overexpression on cell wall chemistry remains unexplored.ResultsWe overexpressed SND2 in Arabidopsis and analyzed homozygous lines with regards to stem chemistry, biomass and fibre secondary cell wall thickness. A line showing upregulation of CesA8 was selected for transcriptome-wide gene expression profiling. We found evidence for upregulation of biosynthetic genes associated with cellulose, xylan, mannan and lignin polymerization in this line, in agreement with significant co-expression of these genes with native SND2 transcripts according to public microarray repositories. Only minor alterations in cell wall chemistry were detected. Transcription factor MYB103, in addition to SND1, was upregulated in SND2-overexpressing plants, and we detected upregulation of genes encoding components of a signal transduction machinery recently proposed to initiate secondary cell wall formation. Several homozygous T4 and hemizygous T1 transgenic lines with pronounced SND2 overexpression levels revealed a negative impact on fibre wall deposition, which may be indirectly attributable to excessive overexpression rather than co-suppression. Conversely, overexpression of SND2 in Eucalyptus stems led to increased fibre cross-sectional cell area.ConclusionsThis study supports a function for SND2 in the regulation of cellulose and hemicellulose biosynthetic genes in addition of those involved in lignin polymerization and signalling. SND2 seems to occupy a subordinate but central tier in the secondary cell wall transcriptional network. Our results reveal phenotypic differences in the effect of SND2 overexpression between woody and herbaceous stems and emphasize the importance of expression thresholds in transcription factor studies.

A thaumatin-like protein gene involved in cotton fiber secondary cell wall development enhances resistance against Verticillium dahliae and other stresses in transgenic tobacco

For the first time, a sea-island cotton (Gossypium barbadense L.) thaumatin-like protein gene (GbTLP1) with a potential role in secondary cell wall development has been overexpressed in tobacco to elucidate its function. The presence of the transgene was verified by Southern blotting and higher expression levels of GbTLP1 in transgenic tobacco plants were revealed by reverse-transcription and quantitative real-time polymerase chain reaction analyses. Transgenic plants with constitutively higher expression of the GbTLP1 showed enhanced resistance against different stress agents, particularly, its performance against Verticillium dahliae was exceptional. Transgenic tobacco plants also exhibited considerable resistance against Fusarium oxysporum and some abiotic stresses including salinity and drought. In this experiment, transgenic plants without GbTLP1 expression were also used as controls, which behaved similar to non-transgenic control plants. Overexpression of GbTLP1 had no significant deleterious effect on plant growth except that flowering was delayed for 3–5weeks. The apparent pleiotropic effect of this novel gene has given us insight to the plant defense mechanism.

Fourier transform infrared spectroscopic approach to the study of the secondary cell wall development in cotton fiber

Cotton fiber maturity is a major yield component and an important fiber quality trait that is directly linked to the quantity of cellulose deposited during the secondary cell wall (SCW) biogenesis. Cotton fiber development consists of five major overlapping stages: differentiation, initiation, polar elongation, secondary cell wall development, and maturation. The transition period between 16 and 21 dpa (days post anthesis) is regarded to represent a major developmental stage between the primary cell wall and the SCW. Fourier Transform Infrared spectroscopy was used to investigate the structural changes that occur during the different developmental stages. The IR spectra of fibers harvested at different stages of development (10, 14, 17, 18, 19, 20, 21, 24, 27, 30, 36, 46, and 56 dpa) show the presence of vibrations located at 1,733 cm−1 (C=O stretching originating from esters or amides) and 1,534 cm−1 (NH2 deformation corresponding to proteins or amino acids). The results converge towards the conclusion that the transition phase between the primary cell wall and the secondary cell wall occurs between 17 and 18 dpa in fibers from TX19 cultivar, while this transition occurs between 21 and 24 dpa in fibers from TX55 cultivar.

Rice BRITTLE CULM 5 (BRITTLE NODE) is Involved in Secondary Cell Wall Formation in the Sclerenchyma Tissue of Nodes

Several brittle culm (bc) mutants known in grasses are considered excellent materials to study the process of secondary cell wall formation. The brittle phenotype of the rice bc5 (brittle node) mutant appears exclusively in the developed nodes, which is distinct from other bc mutants (bc1, 2, 3, 4, 6 and 7) that show the brittle phenotype in culms and leaves. To address the defects of the rice bc5 mutant in node-specific cell wall formation, we analyzed tissue morphology and cell wall composition. The bc5 mutation was found to affect the cell wall deposition of node sclerenchyma tissues at 1 week after heading, the stage at which the cell wall sugar content is reduced, in the bc5 nodes, compared with wild-type nodes. Moreover, decreased accumulation of lignin and thickness of cell walls in the sclerenchyma tissues were also observed in the bc5 nodes. The amounts of cellulose and hemicellulose were reduced to 53 and 65% of those in the wild-type plants, respectively. Sugar composition and glycosidic linkage analyses of the hemicellulose showed that the accumulation of glucuronosyl arabinoxylan in bc5 nodes was perturbed by the mutation. The bc5 locus was narrowed to an approximately 3.1 Mb region of chromosome 2, where none of the other bc genes is located. The bc5 mutation appeared to reduce the expression levels of the OsCesA genes in the nodes after heading. The results indicate that the BC5 gene regulates the development of secondary cell walls of node sclerenchyma tissues.

Overexpression of PwTUA1, a pollen-specific tubulin gene, increases pollen tube elongation by altering the distribution of -tubulin and promoting vesicle transport

Tubulin genes are intimately associated with cell division and cell elongation, which are central to plant secondary cell wall development. However, their roles in pollen tube polar growth remain elusive. Here, a TUA1 gene from Picea wilsonii, which is specifically expressed in pollen, was isolated. Semi-quantitative RT-PCR analysis showed that the amount of PwTUA1 transcript varied at each stage of growth of the pollen tube and was induced by calcium ions and boron. Transient expression analysis in P. wilsonii pollen indicated that PwTUA1 improved pollen germination and pollen tube growth. The pollen of transgenic Arabidopsis overexpressing PwTUA1 also showed a higher percentage of germination and faster growth than wild-type plants not only in optimal germination medium, but also in medium supplemented with elevated levels of exogenous calcium ions or boron. Immunofluorescence and electron microscopy showed α-tubulin to be enriched and more vesicles accumulated in the apex region in germinating transgenic Arabidopsis pollen compared with wild-type plants. These results demonstrate that PwTUA1 up-regulated by calcium ions and boron contributes to pollen tube elongation by altering the distribution of α-tubulin and regulating the deposition of pollen cell wall components during the process of tube growth. The possible role of PwTUA1 in microtubule dynamics and organization was discussed.

Genes Encoding Fasciclin-Like Arabinogalactan Proteins are Specifically Expressed During Cotton Fiber Development

Cotton (Gossypium L.) fiber is a unique experimental system for studying plant cell elongation and cell wall biogenesis. Arabinogalactan proteins (AGPs) are extracellular proteoglycans and play important roles in various aspects of plant growth and development. In the present work, four AGP genes, GhAGP2, GhAGP3, GhAGP4, and GhFLA1, were cloned from cotton fiber through 5′ and 3′ rapid amplification of cDNA ends (RACE). All these genes code for classical AGPs containing N-terminal signal sequence, a central domain rich in Ala, Ser, Thr, Pro/Hyp, and C-terminal hydrophobic transmembrane domain. Additionally all the four putative proteins contain two AGP-like domains, one fasciclin-like domain and a putative C-terminal glycosylphosphatidylinositol lipid anchor signal sequence. Northern blotting analysis with gene-specific probes indicates the transcripts of GhAGP2 and GhFLA1 accumulate in Gossypium hirsutum fiber from 5 to 20 days post anthesis, whereas GhAGP3 and GhAGP4 are abundantly expressed during the developmental stage switching from elongation to the secondary cell wall synthesis. Furthermore, quantitative reverse transcription polymerase chain reaction analysis demonstrates that all these fasciclin-like AGP genes are specifically expressed not only in G. hirsutum fibers but also in Gossypium barbadense fibers. The fasciclin-like domains are typically found in adhesion proteins of animal cell, and the specific expression patterns together with presence of fasciclin-like domains suggest roles for fasciclin-like AGPs in cell-to-cell communication during cotton fiber elongation and secondary cell wall development.