Genome-wide identification, classification and expression analysis of genes encoding putative fasciclin-like arabinogalactan proteins in Chinese cabbage (Brassica rapa L.)

Fasciclin-like arabinogalactan proteins (FLAs), a subclass of arabinogalactan proteins (AGPs), have both predicted AGP-like glycosylated regions and putative fasciclin (FAS) domains, which may function in cell adhesion and communication. Previous studies have identified 21, 27, and 34 FLAs in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and wheat (Triticum aestivum), respectively. In this study, we identified 33 FLAs in the annotated genome of Chinese cabbage (Brassica rapa ssp. pekinensis line Chiifu-401-42). Sequence analysis indicated that FAS domains each contain two highly conserved regions, named H1 and H2, and that 17 FLAs from B. rapa (BrFLAs) possess both of these regions. Prediction of glycosylphosphatidylinositol (GPI) modification sites suggested that 15 BrFLAs were GPI-anchored to the plasma membrane. Additionally, 25 BrFLAs may have been duplicated during the processes that shaped the triplicated genome of the mesopolyploid B. rapa. Expression analyses indicated that BrFLA1, BrFLA11, BrFLA13, BrFLA28 and BrFLA32 were specifically expressed in inflorescence. Meanwhile, BrFLA9 (homologous to AtFLA12) is specifically expressed in stem, and BrFLA6/22 (homologous to AtFLA11) is also highly expressed in stem, suggesting BrFLA6/9/22 may have the same functions as AtFLA11/12 in A. thaliana. Taken together, the identification and bioinformatic analysis of FLAs in B. rapa will open the way for studying their biological functions in plant growth and development as well as evolutionary history of this gene family from A. thaliana to B. rapa.     

Glycosylation of a Fasciclin-Like Arabinogalactan-Protein (SOS5) Mediates Root Growth and Seed Mucilage Adherence via a Cell Wall Receptor-Like Kinase (FEI1/FEI2) Pathway in Arabidopsis

Fundamental processes that underpin plant growth and development depend crucially on the action and assembly of the cell wall, a dynamic structure that changes in response to both developmental and environmental cues. While much is known about cell wall structure and biosynthesis, much less is known about the functions of the individual wall components, particularly with respect to their potential roles in cellular signaling. Loss-of-function mutants of two arabinogalactan-protein (AGP)-specific galactosyltransferases namely, GALT2 and GALT5, confer pleiotropic growth and development phenotypes indicating the important contributions of carbohydrate moieties towards AGP function. Notably, galt2galt5 double mutants displayed impaired root growth and root tip swelling in response to salt, likely as a result of decreased cellulose synthesis. These mutants phenocopy a salt-overly sensitive mutant called sos5, which lacks a fasciclin-like AGP (SOS5/FLA4) as well as a fei1fei2 double mutant, which lacks two cell wall-associated leucine-rich repeat receptor-like kinases. Additionally, galt2gal5 as well as sos5 and fei2 showed reduced seed mucilage adherence. Quintuple galt2galt5sos5fei1fei2 mutants were produced and provided evidence that these genes act in a single, linear genetic pathway. Further genetic and biochemical analysis of the quintuple mutant demonstrated involvement of these genes with the interplay between cellulose biosynthesis and two plant growth regulators, ethylene and ABA, in modulating root cell wall integrity.     

Two Hydroxyproline Galactosyltransferases,GALT5 and GALT2, Function in Arabinogalactan-Protein Glycosylation,Growth and Development in Arabidopsis

Hydroxyproline-O-galactosyltransferase (GALT) initiates O-glycosylation of arabinogalactan-proteins (AGPs). We previously characterized GALT2 (At4g21060), and now report on functional characterization of GALT5 (At1g74800). GALT5 was identified using heterologous expression in Pichia and an in vitro GALT assay. Product characterization showed GALT5 specifically adds galactose to hydroxyproline in AGP protein backbones. Functions of GALT2 and GALT5 were elucidated by phenotypic analysis of single and double mutant plants. Allelic galt5 and galt2 mutants, and particularly galt2 galt5 double mutants, demonstrated lower GALT activities and reductions in β-Yariv-precipitated AGPs compared to wild type. Mutant plants showed pleiotropic growth and development phenotypes (defects in root hair growth, root elongation, pollen tube growth, flowering time, leaf development, silique length, and inflorescence growth), which were most severe in the double mutants. Conditional mutant phenotypes were also observed, including salt-hypersensitive root growth and root tip swelling as well as reduced inhibition of pollen tube growth and root growth in response to β-Yariv reagent. These mutants also phenocopy mutants for an AGP, SOS5, and two cell wall receptor-like kinases, FEI1 and FEI2, which exist in a genetic signaling pathway. In summary, GALT5 and GALT2 function as redundant GALTs that control AGP O-glycosylation, which is essential for normal growth and development.     

Fucosylated arabinogalactan-proteins are required for full root cell elongation in arabidopsis

The Arabidopsis thaliana mutant mur1 is affected in the biosynthesis of l-fucose and has less than 2% of the normal amounts of this sugar in the cell walls of its aerial parts. Although in roots the reduction of l-fucose is only 40%, this causes a decrease of about 50% in root cell elongation. Since arabinogalactan-proteins (AGPs) are known to play a role in plant cell expansion we studied the composition of mur1 root AGPs. Arabidopsis root AGPs were shown to contain l-fucose, which was reduced in level in mur1 AGPs. In wild-type plants, an l-fucose containing epitope is present in AGPs in the cell wall of differentiating root cells. Addition of eel lectin, which specifically recognizes this epitope, and not fucose in other wall polymers, can phenocopy mur1 roots. Several lines of evidence are presented to support the contention that l-fucose containing root AGPs are required for the full elongation of root cells.     

Genetic Interactions at the Fla10 Locus: Suppressors and Synthetic Phenotypes That Affect the Cell Cycle and Flagellar Function in Chlamydomonas Reinhardtii

Through the isolation of suppressors of temperature-sensitive flagellar assembly mutations at the FLA10 locus of Chlamydomonas reinhardtii, we have identified six other genes involved in flagellar assembly. Mutations at these suppressor loci, termed SUF1-SUF6, display allele specificity with respect to which fla10- mutant alleles they suppress. An additional mutation, apm1-122, which confers resistance to the plant herbicides amiprophos-methyl and oryzalin, was also found to interact with mutations at the FLA10 locus. The apm1-122 mutation in combination with three fla10- mutant alleles results in synthetic cold-sensitive cell division defects, and in combination with an additional pseudo-wild-type fla10- allele yields a synthetic temperature-sensitive flagellar motility phenotype. Based upon the genetic interactions of these loci, we propose that the FLA10 gene product interacts with multiple components of the flagellar apparatus and plays a role both in flagellar assembly and in the cell cycle.     

The Chlamydomonas FLA10 gene encodes a novel kinesin-homologous protein

Many genes on the uni linkage group of Chlamydomonas affect the basal body/flagellar apparatus. Among these are five FLA genes, whose mutations cause temperature-sensitive defects in flagellar assembly. We present the molecular analysis of a gene which maps to fla10 and functionally rescues the fla10 phenotype. Nucleotide sequencing revealed that the gene encodes a kinesin-homologous protein, KHP1. The 87-kD predicted KHP1 protein, like kinesin heavy chain, has an amino- terminal motor domain, a central alpha-helical stalk, and a basic, globular carboxy-terminal tail. Comparison to other kinesin superfamily members indicated striking similarity (64% identity in motor domains) to a mouse gene, KIF3, expressed primarily in cerebellum. In synchronized cultures, the KHP1 mRNA accumulated after cell division, as did flagellar dynein mRNAs. KHP1 mRNA levels also increased following deflagellation. Polyclonal antibodies detected KHP1 protein in Western blots of purified flagella and axonemes. The protein was partially released from axonemes with ATP treatment, but not with AMP- PNP. Western blot analysis of axonemes from various motility mutants suggested that KHP1 is not a component of radial spokes, dynein arms, or the central pair complex. The quantity of KHP1 protein in axonemes of the mutant fla10-1 was markedly reduced, although no reduction was observed in two other uni linkage group mutants, fla9 and fla11. Furthermore, fla10-1 was rescued by transformation with KHP1 genomic DNA. These results indicate that KHP1 is the gene product of FLA10 and suggest a novel role for this kinesin-related protein in flagellar assembly and maintenance.     

Target of rapamycin regulates development and ribosomal RNA expression through kinase domain in Arabidopsis

Target of rapamycin (TOR) is a central regulator of cell growth, cell death, nutrition, starvation, hormone, and stress responses in diverse eukaryotes. However, very little is known about TOR signaling and the associated functional domains in plants. We have taken a genetic approach to dissect TOR functions in Arabidopsis (Arabidopsis thaliana) and report here that the kinase domain is essential for the role of TOR in embryogenesis and 45S rRNA expression. Twelve new T-DNA insertion mutants, spanning 14.2 kb of TOR-encoding genomic region, have been characterized. Nine of these share expression of defective kinase domain and embryo arrest at 16 to 32 cell stage. However, three T-DNA insertion lines affecting FATC domain displayed normal embryo development, indicating that FATC domain was dispensable in Arabidopsis. Genetic complementation showed that the TOR kinase domain alone in tor-10/tor-10 mutant background can rescue early embryo lethality and restore normal development. Overexpression of full-length TOR or kinase domain in Arabidopsis displayed developmental abnormalities in meristem, leaf, root, stem, flowering time, and senescence. We further show that TOR, especially the kinase domain, plays a role in ribosome biogenesis by activating 45S rRNA production. Of the six putative nuclear localization sequences in the kinase domain, nuclear localization sequence 6 was identified to confer TOR nuclear targeting in transient expression assays. Chromatin immunoprecipitation studies revealed that the HEAT repeat domain binds to 45S rRNA promoter and the 5' external transcribed spacer elements motif. Together, these results show that TOR controls the embryogenesis, postembryonic development, and 45S rRNA production through its kinase domain in Arabidopsis.     

DEAP1 encodes a fasciclin-like arabinogalactan protein required for male fertility in rice

Arabinogalactan proteins(AGPs) are widely distributed in plant cells. Fasciclin-like AGPs(FLAs)belong to a subclass of AGPs that play important roles in plant growth and development. However,little is known about the biological functions of rice FLA. Herein, we report the identification of a male-sterile mutant of DEFECTIVE EXINE AND APERTURE PATTERNING1(DEAP1) in rice. The deap1 mutant anthers produced aberrant pollen grains with defective exine formation and a flattened aperture annulus and ex...     

The reb1-1 mutation of Arabidopsis alters the morphology of trichoblasts,the expression of arabinogalactan-proteins and the organization of cortical microtubules

The root epidermal bulger 1 ( reb1) mutant of Arabidopsis thaliana (L.) Heynh. is characterized by a reduced elongation rate of the primary root and by the bulging of many, but not all, root epidermal cells. In this study, we investigated cell wall structure of root epidermal cells in reb1-1 by using serial sectioning, and light and electron microscopy in combination with immuno-cytochemistry and polysaccharide staining. We found that: (i) Cell bulging in the mutant was initiated in the zone of elongation of the root, and occurred exclusively in trichoblasts. (ii) reb1-1 and wild-type root cells stained identically with anti-pectin antibodies, such as JIM5. In contrast, the anti-arabinogalactan-protein antibodies, JIM14 and LM2, stained all epidermal cells in the wild type and trichoblasts preferentially, but in reb1-1 they stained the atrichoblasts only. (iii) Compared to the wild type, mutant trichoblasts had a thinner outer epidermal cell wall, which presented abnormal periodic acid-thio carbohydrazide silver proteinate (PATAg) staining. In addition, we investigated the organization of cortical microtubules in a reb1-1 mutant line expressing a green-fluorescent protein fused to a microtubule-binding domain from human microtubule-associated protein 4. Microtubules in the swollen trichoblasts of reb1-1 were either disordered or absent entirely. Together our findings indicate that the reb1-1 mutation results in an abnormal trichoblast cell wall, and suggest that cell surface arabinogalactan-proteins are required for anisotropic expansion and for orienting cortical microtubules.     

DAWDLE, a forkhead-associated domain gene, regulates multiple aspects of plant development

Phosphoprotein-binding domains are found in many different proteins and specify protein-protein interactions critical for signal transduction pathways. Forkhead-associated (FHA) domains bind phosphothreonine and control many aspects of cell proliferation in yeast (Saccharomyces cerevisiae) and animal cells. The Arabidopsis (Arabidopsis thaliana) protein kinase-associated protein phosphatase includes a FHA domain that mediates interactions with receptor-like kinases, which in turn regulate a variety of signaling pathways involved in plant growth and pathogen responses. Screens for insertional mutations in other Arabidopsis FHA domain-containing genes identified a mutant with pleiotropic defects. dawdle (ddl) plants are developmentally delayed, produce defective roots, shoots, and flowers, and have reduced seed set. DDL is expressed in the root and shoot meristems and the reduced size of the root apical meristem in ddl plants suggests a role early in organ development.     

AtAGP30, an arabinogalactan-protein in the cell walls of the primary root, plays a role in root regeneration and seed germination

Arabinogalactan-proteins (AGPs) are extracellular proteoglycans that are implicated in many plant growth and developmental processes, but in no case has a biological function been assigned to a particular AGP. AtAGP30 is a non-classical AGP core protein from Arabidopsis that is expressed only in roots. Analysis of the corresponding mutant, agp30, has revealed that the wild-type gene product is required in vitro for root regeneration and in planta for the timing of seed germination. The mutant shows a suppression of the abscisic acid (ABA)-induced delay in germination and altered expression of some ABA-regulated genes. This suggests that AtAGP30 functions in the ABA response. By analogy to proteoglycan-mediated regulation of growth-factor-signalling pathways in animals, our data indicate that phytohormone activity in plants can be modulated by AGPs.     

Expression of AtPRP3, a proline-rich structural cell wall protein from Arabidopsis, is regulated by cell-type-specific developmental pathways involved in root hair formation

The tightly regulated expression patterns of structural cell wall proteins in several plant species indicate that they play a crucial role in determining the extracellular matrix structure for specific cell types. We demonstrate that AtPRP3, a proline-rich cell wall protein in Arabidopsis, is expressed in root-hair-bearing epidermal cells at the root/shoot junction and within the root differentiation zone of light-grown seedlings. Several lines of evidence support a direct relationship between AtPRP3 expression and root hair development. AtPRP3/beta-glucuronidase (GUS) expression increased in roots of transgenic seedlings treated with either 1-aminocyclopropane-1-carboxylic acid (ACC) or alpha-naphthaleneacetic acid (alpha-NAA), compounds known to promote root hair formation. In the presence of 1-alpha-(2-aminoethoxyvinyl)glycine (AVG), an inhibitor of ethylene biosynthesis, AtPRP3/GUS expression was strongly reduced, but could be rescued by co-addition of ACC or alpha-NAA to the growth medium. In addition, AtPRP3/GUS activity was enhanced in ttg and gl2 mutant backgrounds that exhibit ectopic root hairs, but was reduced in rhd6 and 35S-R root-hair-less mutant seedlings. These results indicate that AtPRP3 is regulated by developmental pathways involved in root hair formation, and are consistent with AtPRP3's contributing to cell wall structure in Arabidopsis root hairs.     

Short-Root regulates primary, lateral, and adventitious root development in Arabidopsis

Short-Root (SHR) is a well-characterized regulator of radial patterning and indeterminacy of the Arabidopsis (Arabidopsis thaliana) primary root. However, its role during the elaboration of root system architecture remains unclear. We report that the indeterminate wild-type Arabidopsis root system was transformed into a determinate root system in the shr mutant when growing in soil or agar. The root growth behavior of the shr mutant results from its primary root apical meristem failing to initiate cell division following germination. The inability of shr to reactivate mitotic activity in the root apical meristem is associated with the progressive reduction in the abundance of auxin efflux carriers, PIN-FORMED1 (PIN1), PIN2, PIN3, PIN4, and PIN7. The loss of primary root growth in shr is compensated by the activation of anchor root primordia, whose tissues are radially patterned like the wild type. However, SHR function is not restricted to the primary root but is also required for the initiation and patterning of lateral root primordia. In addition, SHR is necessary to maintain the indeterminate growth of lateral and anchor roots. We conclude that SHR regulates a wide array of Arabidopsis root-related developmental processes.     

Identification of three potent hydroxyproline O‐galactosyltransferases in Arabidopsis

Arabinogalactan proteins (AGPs) are plant‐specific extracellular glycoproteins implicated in a variety of processes during growth and development. AGP biosynthesis involves O‐galactosylation of hydroxyproline (Hyp) residues followed by a stepwise elongation of the complex sugar chains. However, functionally dominant Hyp O‐galactosyltransferases, such that their disruption produces phenocopies of AGP‐deficient mutants, remain to be identified. Here, we purified and identified three potent Hyp O‐galactosyltransferases, HPGT1, HPGT2 and HPGT3, from Arabidopsis microsomal fractions. Loss‐of‐function analysis indicated that approximately 90% of the endogenous Hyp O‐galactosylation activity is attributable to these three enzymes. AGP14 expressed in the triple mutant migrated much faster on SDS‐PAGE than when expressed in wild‐type, confirming a considerable decrease in levels of glycosylation of AGPs in the mutant. Loss‐of‐function mutant plants exhibited a pleiotropic phenotype of longer lateral roots, longer root hairs, radial expansion of the cells in the root tip, small leaves, shorter inflorescence stems, reduced fertility and shorter siliques. Our findings provide genetic evidence that Hyp‐linked arabinogalactan polysaccharide chains are critical for AGP function and clues to how arabinogalactan moieties of AGPs contribute to cell‐to‐cell communication during plant growth and development.