Morphological,genetic and molecular characteristics of barley root hair mutants

Root hairs are tubular outgrowths of specialized epidermal cells called trichoblasts. They affect anchoring plants in soil, the uptake of water and nutrients and are the sites of the interaction between plants and microorganisms. Nineteen root hair mutants of barley representing different stages of root hair development were subjected to detailed morphological and genetic analyses. Each mutant was monogenic and recessive. An allelism test revealed that nine loci were responsible for the mutated root hair phenotypes in the collection and 1–4 mutated allelic forms were identified at each locus. Genetic relationships between the genes responsible for different stages of root hair formation were established. The linkage groups of four loci rhl1, rhp1, rhi1 and rhs1, which had previously been mapped on chromosomes 7H, 1H, 6H and 5H, respectively, were enriched with new markers that flank the genes at a distance of 0.16 cM to 4.6 cM. The chromosomal position of three new genes – two that are responsible for the development of short root hairs (rhs2 and rhs3) and the gene that controls an irregular root hair pattern (rhi2) – were mapped on chromosomes 6H, 2H and 1H, respectively. A comparative analysis of the agrobotanical parameters between some mutants and their respective parental lines showed that mutations in genes responsible for root hair development had no effect on the agrobotanical performance of plants that were grown under controlled conditions. The presented mutant collection is a valuable tool for further identification of genes controlling root hair development in barley.     

Analyses of mutants of three genes that influence root hair development in Zea mays (Gramineae) suggest that root hairs are dispensable

Root hairs are specialized epidermal cells that are thought to play an important role in plant nutrition by facilitating the absorption of water and nutrients. Three maize mutants with abnormal root hair morphologies (rthl, rth2, and rth3) have been isolated from Mutator transposon stocks. All three root hair mutant phenotypes are controlled by single recessive alleles. The rthl mutant initiates normal-looking root hair primordia that fail to elongate. The normal-looking root hair primordia of the rth2 mutant elongate to only approximately one-fifth to one-fourth the length of wild type root hairs. Like rth1 primordia, rth3 primordia undergo little elongation. However, unlike the relatively normal-looking rth1 primordia, rth3 primordia are distinctly abnormal when viewed through a scanning electron microscope. The rth1 mutant exhibits pleiotropic nutrient deficiencies, while the rth2 and rth3 mutants grow vigorously. This finding suggests that under some environmental conditions, root hairs are less important to plant growth than has been previously thought. The rthl, rth2, and rth3 genes have been mapped to chromosomes 1L, 5L, and 1S, respectively, via crosses with BA translocation stocks. The rth2 allele exhibits reduced transmission through the male gametophyte, but a normal rate of transmission through female gametophytes; rth1 and rth3 are transmitted at normal rates.     

Isolation and initial characterization of temperature-sensitive mutants of Arabidopsis thaliana that are impaired in root redifferentiation

As tools for studying adventitious organogenesis, three temperature-sensitive mutants of Arabidopsis thaliana, designated rrd1, rrd2, and rrd4, were isolated by screening with callus-mediated root redifferentiation from hypocotyl explants as an index phenotype. Phenotypes of these mutants were characterized with special regard to their tissue-culture responses. The obtained results suggest that the RRD1 and RRD2 genes participate in some fundamental processes required for active cell proliferation and that the RRD4 gene is involved in the acquisition step of competence for cell proliferation during callus initiation in hypocotyl explants.     

Reorganization and in vivo dynamics of microtubules during Arabidopsis root hair development

Root hairs emerge from epidermal root cells (trichoblasts) and differentiate by highly localized tip growth. Microtubules (MTs) are essential for establishing and maintaining the growth polarity of root hairs. The current knowledge about the configuration of the MT cytoskeleton during root hair development is largely based on experiments on fixed material, and reorganization and in vivo dynamics of MTs during root hair development is at present unclear. This in vivo study provides new insights into the mechanisms of MT (re)organization during root hair development in Arabidopsis (Arabidopsis thaliana). Expression of a binding site of the MT-associated protein-4 tagged with green fluorescent protein enabled imaging of MT nucleation, growth, and shortening and revealed distinct MT configurations. Depending on the dynamics of the different MT populations during root hair development, either repeated two-dimensional (x, y, t) or repeated three-dimensional (x, y, z, t) scanning was performed. Furthermore, a new image evaluation tool was developed to reveal important data on MT instability. The data show how MTs reorient after apparent contact with other MTs and support a model for MT alignment based on repeated reorientation of dynamic MT growth.     

Arabinogalactan proteins during the development of soybean root nodules

In soybean (Glycine max (L.) Merr.) root nodules the level of hydroxyproline-containing molecules is developmentally regulated. Hydroxyproline accumulates in both nodule cortex and medulla. In the cortex, the hydroxyproline is mainly localized in the cell wall, presumably as extensin, but in the medulla it is mainly in the soluble fraction as an arabinogalactan protein (AGP). Nodule-specific AGPs are present at early nodulation. The highest concentration of AGP is in the nodule medulla, followed by nodule cortex, uninfected roots, leaves, flowers, pods and seeds. Root nodules and all organs of the soybean plant that were tested were found to express a tissue-specific set of arabinogalactan proteins.Abbreviation AGP Arabinogalactan protein     

Isolation and characterization of cDNA clones corresponding with mRNAs that accumulate during auxin-induced lateral root formation

Lateral root formation in root cultures of Arabidopsis thaliana can be initiated by exogenous addition of auxin. In order to find cDNA clones of which the corresponding mRNAs accumulate during this process, a cDNA library was constructed from root cultures treated with the active auxin 1-naphthaleneacetic acid (1- NAA). Differential screening of this library with cDNA probes derived from mRNA populations isolated from root cultures treated with 1-NAA and the inactive analogue 2-naphthaleneacetic acid (2-NAA) led to the isolation of four cDNA clones, designated AIR1, AIR3, AIR9 and AIR12. Accumulation of the mRNAs starts between 4 and 8 h and continues till at least 24 h after addition of an active auxin. Sequence analysis revealed that AIR1 encodes a protein that is related to a large family of proteins that consist of a proline-rich or glycine- rich N-terminus and a hydrophobic, possibly membrane spanning C- terminus. The putative function of these proteins is coupling of the cell wall to the plasma membrane. Surprisingly, AIR1 lacks the proline-rich or glycine-rich N-terminus which is thought to be important for interaction with the cell wall. AIR3 encodes a subtilisin-like serine protease which is believed to be active outside the plant cell. Although AIR9 and AIR12 do not show any significant homology to sequences in the database, they are also predicted to function outside the cell. Our screening thus indicates that a variety of genes encoding extracellular proteins are activated during auxin-induced lateral root formation.     

Characterisation and functional analysis of two barley caleosins expressed during barley caryopsis development

Two full-length cDNA sequences homologous to caleosin, a seed-storage oil-body protein from sesame, were identified from a series of barley grain development cDNA libraries and further characterised. The cDNAs, subsequently termed HvClo1 and HvClo2, encode proteins of 34 kDa and 28 kDa, respectively. Real-time RT-PCR indicated that HvClo1 is expressed abundantly during the later stages of embryogenesis and is seed-specific, accumulating in the scutellum of mature embryos. HvClo2 is expressed mainly in the endosperm tissues of the developing grain. We show that HvClo1 and HvClo2 are paralogs that co-segregate on barley chromosome 2HL. Transient expression of HvClo1 in lipid storage and non-storage cells of barley using biolistic particle bombardment indicates that caleosins have different subcellular locations from the structural oil-body protein oleosin, and by inference participate in different sorting pathways. We observe that caleosin sorts via small vesicles, suggesting a likely association with lipid trafficking, membrane expansion and oil-body biogenesis.     

The seminal root primordia in barley and the participation of their non-meristematic cells in root construction

In addition to the primary seminal primordium, the so-called secondary seminal root primordia are also initiated in a barley embryo. The primary root primordium is developmentally most advanced. It is formed by root meristem covered with the root cap, and by a histologically determined region with completed cell division. On germination, the restoration of growth processes begins in this non-meristematic region of root primordium by cell elongation, with the exception of the zone adjacent to the scutellar node, the cells of which do not elongate but continue differentiating. In the root primordia initiated later, the zone with completed cell division is relatively shorter, in the youngest primordia the non-meristematic cells may be lacking. The root meristem is reactivated after the primary root primordium has broken through the sheath-like coleorrhiza and emerges from the caryopsis as the primary root. The character of root meristem indicates a reduced water content at the embryonic development of root primordium. With progressing growth the root apex becomes thinner, the meristematic region becomes longer, and the differences in the extent of cell division between individual cell types increase. — The primary root base is formed of cells pre-existing in the seminal root primordium. Upon desiccation of caryopsis in maturation, and subsequent quiescent period, their development was temporarily broken, proceeding with the onset of germination. The length of this postembryonically non-dividing basal zone is different in individual cell types. The column of central metaxylem characteristic of the smallest number of cell cycles, has, under the given conditions, a mean length of about 22 mm, whereas the pericycle, as the tissue with most prolonged cell division, has a mean length of about 6 mm. In the seminal root primordia initiated later the non-dividing areas are relatively shorter. The basal region of seminal roots thus differs in its ontogenesis from the increase which is formed “de novo” by the action of root meristem upon seed germination.     

Germ cell death and their removal during initial stages of testicular ischemia and cryptorchidism: a comparative analysis

Germ cell death and their removal from the seminiferous epithelium are common in the affected testis in conditions of unilateral ischemia or cryptorchidism; the similarities and differences, however, have not been studied between these two conditions. The present study was designed to examine the severity of the effect on testicular germ cells during the initial stages of both ischemia and cryptorchidism, which have significant implications on the restoration of fertility following surgical repair. Complete absence of spermatids was observed following 12 hr of ischemia as compared to 7 days of cryptorchidism. Germ cell removal in either case was in the direction of lumen to basement membrane leaving only a single layer of cells by 24 hr of unilateral ischemia as compared to 15 days of cryptorchidism. Levels of intratesticular testosterone was found lower in cryptorchidism (7 days) but not in ischemia till 24 hrs. Giant cells frequently observed in cryptorchid testis were absent in the ischemic seminiferous epithelium. There was a gradual increase in the number of apoptotic and non-viable cells; the latter was more than 95% by 24 hr of ischemia. In contrast, approximately 85% testicular cells were nonviable till 15 days of cryptorchidism. The 1c peak representing the population of haploid cells was significantly reduced in cryptorchidism (7 days), while the peak was completely abolished by 24 hr of ischemia. Rise in the levels of oxidative stress in the affected testis was observed identically during the initial stages. These findings indicate that coupled with the rise in tissue oxidative stress, the number of apoptotic/nonviable germ cells was alarmingly high (> 80%) by 15 days of cryptochidism or 24 hr of ischemia. Restoration of complete spermatogenesis following surgical repair may not be possible in such cases because of these acute adverse effects.     

Hormones act downstream of TTG and GL2 to promote root hair outgrowth during epidermis development in the Arabidopsis root.

The Arabidopsis root produces a position-dependent pattern of hair-bearing and hairless cell types during epidermis development. Five loci (TRANSPARENT TESTA GLABRA [TTG], GLABRA2 [GL2], ROOT HAIR DEFECTIVE6 [RHD6], CONSTITUTIVE TRIPLE RESPONSE1 [CTR1], and AUXIN RESISTANT2 [AXR2]) and the plant hormones ethylene and auxin have been reported to affect the production of root hair and hairless cells in the Arabidopsis root. In this study, genetic, molecular, and physiological tests were employed to define the roles of these loci and hormones. Epistasis tests and reporter gene studies indicated that the hairless cell-promoting genes TTG and GL2 are likely to act early to negatively regulate the ethylene and auxin pathways. Studies of the developmental timing of the hormone effects indicated that ethylene and auxin pathways promote root hair outgrowth after cell-type differentiation has been initiated. The genetic analysis of ethylene-and auxin-related mutations showed that root hair formation is influenced by a network of hormone pathways, including a partially redundant ethylene signaling pathway. A model is proposed in which the patterning of root epidermal cells in Arabidopsis is regulated by the cell position-dependent action of the TTG/GL2 pathway, and the ethylene and auxin hormone pathways act to promote root hair outgrowth at a relatively late stage of differentiation.     

Arabidopsis mutants in short- and medium-chain acyl-CoA oxidase activities accumulate acyl-CoAs and reveal that fatty acid beta-oxidation is essential for embryo development

The short-chain acyl-CoA oxidase (ACX4) is one of a family of ACX genes that together catalyze the first step of peroxisomal fatty acid beta-oxidation during early, postgerminative growth in oilseed species. Here we have isolated and characterized an Arabidopsis thaliana mutant containing a T-DNA insert in ACX4. In acx4 seedlings, short-chain acyl-CoA oxidase activity was reduced by greater than 98%, whereas medium-chain activity was unchanged from wild type levels. Despite the almost complete loss of short-chain activity, lipid catabolism and seedling growth and establishment were unaltered in the acx4 mutant. However, the acx4 seedlings accumulated high levels (31 mol %) of short-chain acyl-CoAs and showed resistance to 2,4-dichlorophenoxybutyric acid, which is converted to the herbicide and auxin analogue 2,4-dichlorophenoxyacetic acid by beta-oxidation. A mutant in medium-chain length acyl-CoA activity (acx3) (1) shows a similar phenotype to acx4, and we show here that acx3 seedlings accumulate medium-chain length acyl-CoAs (16.4 mol %). The acx3 and acx4 mutants were crossed together, and remarkably, the acx3acx4 double mutants aborted during the first phase of embryo development. We propose that acx3acx4 double mutants are nonviable because they have a complete block in short-chain acyl-CoA oxidase activity. This is the first demonstration of the effects of eliminating (short-chain) beta-oxidation capacity in plants and shows that a functional beta-oxidation cycle is essential in the early stages of embryo development.     

Physiology and proteome responses of two contrasting rice mutants and their wild type parent under salt stress conditions at the vegetative stage

Salinity is one of the major environmental limiting factors that affects growth and productivity of rice (Oryza sativa L.) worldwide. Rice is among the most sensitive crops to salinity, especially at early vegetative stages. In order to get a better understanding of molecular pathways affected in rice mutants showing contrasting responses to salinity, we exploited the power of 2-DE based proteomics to explore the proteome changes associated with salt stress response. Our physiological observations showed that standard evaluation system (SES) scores, Na⁺ and K⁺ concentrations in shoots and Na⁺/K⁺ ratio were significantly different in contrasting mutants under salt stress condition. Proteomics analysis showed that, out of 854 protein spots which were reproducibly detected, 67 protein spots showed significant responses to salt stress. The tandem mass spectrometry analysis of these significantly differentially accumulated proteins resulted in identification of 34 unique proteins. These proteins are involved in various molecular processes including defense to oxidative stresses, metabolisms, photosynthesis, protein synthesis and processing, signal transduction. Several of the identified proteins were emerged as key participants in salt stress tolerance. The possible implication of salt responsive proteins in plant adaptation to salt stress is discussed.     

A simplified method for the electrophoretic analysis of hair proteins

Hair proteins have been analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis using a simplified method without S-carboxymethylation, protein fractionation and lyophilization. The molecular weights of the proteins have been determined and human, rat, guinea pig, rabbit, gerbil, cow and sheep hair compared. These molecular weight values are consistent with those obtained using physical methods as compared to anomalously high values previously reported following electrophoresis of S-carboxymethylated hair proteins. Additional high molecular weight proteins, possibly of a dimeric nature, have also been detected.     

Root, root hair, and symbiotic mutants of the model legume Lotus japonicus

To gain an overview of plant factors controlling nodule number and organogenesis, an extensive screening using model legume Lotus japonicus was carried out. This screening involved 40,000 M2 seeds, and 32 stable mutant lines were isolated. From these, 16 mutant lines maintaining the phenotypic variation were selected and genetically analyzed. With respect to nodule number, four loci were identified, Ljsym77, Ljsym78, slippery root (slp), and radial organization1 (rdo1). The former two mutants have an increased number of nodules, while the latter two have a decreased number. Ljsym78-1 and Ljsym78-2 are hypernodulating mutants with a branched root system and were found to be allelic to Ljsym16. The phenotype of the Ljsym77 mutant was highly pleiotropic, being deficient in light and gravity responses. The slp mutant was isolated as a low-nodulating mutant lacking root hairs. Concerning nodule organogenesis, nine symbiotic loci were identified, including the two loci alb1 and fen1. Mutants affecting the developmental process of nodule organogenesis were placed in three phenotypic categories: Nod- (Ljsym70 to Ljsym73), Hist- (alb1-1, alb1-2, and Ljsym79), and Fix- (fen1, Ljsym75, and Ljsym81).