Rosemary G. White

Gibberellin Signaling in Barley Aleurone Cells. Control of SLN1 and GAMYB Expression

We have previously identified GAMYB, a gibberellin (GA)-regulated transcriptional activator of α-amylase gene expression, in aleurone cells of barley (Hordeum vulgare). To examine the regulation of GAMYB expression, we describe the use of nuclear run-on experiments to show that GA causes a 2-fold increase in the rate of GAMYB transcription and that the effect of GA can be blocked by abscisic acid (ABA). To identify GA-signaling components that regulate GAMYB expression, we examined the role of SLN1, a negative regulator of GA signaling in barley. SLN1, which is the product of the Sln1(Slender1) locus, is necessary for repression of GAMYB in barley aleurone cells. The activity of SLN1 in aleurone cells is regulated posttranslationally. SLN1 protein levels decline rapidly in response to GA before any increase in GAMYB levels. Green fluorescent protein-SLN1 fusion protein was targeted to the nucleus of aleurone protoplasts and disappeared in response to GA. Evidence from a dominant dwarf mutant at Sln1, and from thegse1 mutant (that affects GA “sensitivity”), indicates that GA acts by regulating SLN1 degradation and not translation. Mutation of the DELLA region of SLN1 results in increased protein stability in GA-treated layers, indicating that the DELLA region plays an important role in GA-induced degradation of SLN1. Unlike GA, ABA had no effect on SLN1 stability, confirming that ABA acts downstream of SLN1 to block GA signaling.

Genes encoding proteins of the cation diffusion facilitator family that confer manganese tolerance.

The yeast Saccharomyces cerevisiae expressing a cDNA library prepared from Stylosanthes hamata was screened for enhanced Mn2+ tolerance. From this screen, we identified four related cDNAs that encode membrane-bound proteins of the cation diffusion facilitator (CDF) family. One of these cDNAs (ShMTP1) was investigated in detail and found to confer Mn2+ tolerance to yeast by internal sequestration rather than by efflux of Mn2+. Expression of ShMTP1 in a range of yeast mutants suggested that it functions as a proton:Mn2+ antiporter on the membrane of an internal organelle. Similarly, when expressed in Arabidopsis, ShMTP1 conferred Mn2+ tolerance through internal sequestration. The ShMTP1 protein fused to green fluorescent protein was localized to the tonoplast of Arabidopsis cells but appeared to localize to the endoplasmic reticulum of yeast. We suggest that the ShMTP1 proteins are members of the CDF family involved in conferring Mn2+ tolerance and that at least one of these proteins (ShMTP1) confers tolerance by sequestering Mn2+ into internal organelles.

The distribution and abundance of wheat roots in a dense, structured subsoil – implications for water uptake

We analysed the abundance, spatial distribution and soil contact of wheat roots in dense, structured subsoil to determine whether incomplete extraction of subsoil water was due to root system limitations. Intact soil cores were collected to 1.6 m below wheat crops at maturity on a red Kandosol in southern Australia. Wheat roots, remnant roots, soil pores and root-soil contact were quantified at fresh breaks in the soil cores. In surface soil layers (<0.6 m) 30-40% of roots were clumped within pores and cracks in the soil, increasing to 85-100% in the subsoil (>0.6 m), where 44% of roots were in pores with at least three other roots. Most pores contained no roots, with occupancy declining from 20% in surface layers to 5% in subsoil. Wheat roots clumped into pores contacted the surrounding soil via numerous root hairs, whereas roots in cracks were appressed to the soil surface and had very few root hairs. Calculations assuming good root-soil contact indicated that root density was sufficient to extract available subsoil water, suggesting that uptake is constrained at the root-soil interface. To increase extraction of subsoil water, genetic targets could include increasing root-soil contact with denser root hairs, and increasing root proliferation to utilize existing soil pores.

The MicroRNA159-Regulated GAMYB-like Genes Inhibit Growth and Promote Programmed Cell Death in Arabidopsis

The microRNA159 (miR159) family represses the conserved GAMYB-like genes that encode R2R3 MYB domain transcription factors that have been implicated in gibberellin (GA) signaling in anthers and germinating seeds. In Arabidopsis (Arabidopsis thaliana), the two major miR159 family members, miR159a and miR159b, are functionally specific for two GAMYB-like genes, MYB33 and MYB65. These transcription factors have been shown to be involved in anther development, but there are differing reports about their role in the promotion of flowering and little is known about their function in seed germination. To understand the function of this pathway, we identified the genes and processes controlled by these GAMYB-like genes. First, we demonstrate that miR159 completely represses MYB33 and MYB65 in vegetative tissues. We show that GA does not release this repression and that these transcription factors are not required for flowering or growth. By contrast, in the absence of miR159, the deregulation of MYB33 and MYB65 in vegetative tissues up-regulates genes that are highly expressed in the aleurone and GA induced during seed germination. Confirming that these genes are GAMYB-like regulated, their expression was reduced in myb33.myb65.myb101 seeds. Aleurone vacuolation, a GA-mediated programmed cell death process required for germination, was impaired in these seeds. Finally, the deregulation of MYB33 and MYB65 in vegetative tissues inhibits growth by reducing cell proliferation. Therefore, we conclude that miR159 acts as a molecular switch, only permitting the expression of GAMYB-like genes in anthers and seeds. In seeds, these transcription factors participate in GA-induced pathways required for aleurone development and death.

Control of Abscisic Acid Catabolism and Abscisic Acid Homeostasis Is Important for Reproductive Stage Stress Tolerance in Cereals

Drought stress at the reproductive stage causes pollen sterility and grain loss in wheat (Triticum aestivum). Drought stress induces abscisic acid (ABA) biosynthesis genes in anthers and ABA accumulation in spikes of drought-sensitive wheat varieties. In contrast, drought-tolerant wheat accumulates lower ABA levels, which correlates with lower ABA biosynthesis and higher ABA catabolic gene expression (ABA 8′-hydroxylase). Wheat TaABA8′OH1 deletion lines accumulate higher spike ABA levels and are more drought sensitive. ABA treatment of the spike mimics the effect of drought, causing high levels of sterility. ABA treatment represses the anther cell wall invertase gene TaIVR1, and drought-tolerant lines appeared to be more sensitive to the effect of ABA. Drought-induced sterility shows similarity to cold-induced sterility in rice (Oryza sativa). In cold-stressed rice, the rate of ABA accumulation was similar in cold-sensitive and cold-tolerant lines during the first 8 h of cold treatment, but in the tolerant line, ABA catabolism reduced ABA levels between 8 and 16 h of cold treatment. The ABA biosynthesis gene encoding 9-cis-epoxycarotenoid dioxygenase in anthers is mainly expressed in parenchyma cells surrounding the vascular bundle of the anther. Transgenic rice lines expressing the wheat TaABA8′OH1 gene under the control of the OsG6B tapetum-specific promoter resulted in reduced anther ABA levels under cold conditions. The transgenic lines showed that anther sink strength (OsINV4) was maintained under cold conditions and that this correlated with improved cold stress tolerance. Our data indicate that ABA and ABA 8′-hydroxylase play an important role in controlling anther ABA homeostasis and reproductive stage abiotic stress tolerance in cereals.

Genotypic and developmental evidence for the role of plasmodesmatal regulation in cotton fiber elongation mediated by callose turnover.

Cotton fibers are single-celled hairs that elongate to several centimeters long from the seed coat epidermis of the tetraploid species (Gossypium hirsutum and Gossypium barbadense). Thus, cotton fiber is a unique system to study the mechanisms of rapid cell expansion. Previous work has shown a transient closure of plasmodesmata during fiber elongation (Y.-L. Ruan, D.J. Llewellyn, R.T. Furbank [2001] Plant Cell 13: 47–60). To examine the importance of this closure in fiber elongation, we compared the duration of the plasmodesmata closure among different cotton genotypes differing in fiber length. Confocal imaging of the membrane-impermeant fluorescent molecule carboxyfluorescein revealed a genotypic difference in the duration of the plasmodesmata closure that positively correlates with fiber length among three tetraploid genotypes and two diploid progenitors. In all cases, the closure occurred at the rapid phase of elongation. Aniline blue staining and immunolocalization studies showed that callose deposition and degradation at the fiber base correlates with the timing of plasmodesmata closure and reopening, respectively. Northern analyses showed that the expression of a fiber-specific β-1,3-glucanase gene, GhGluc1, was undetectable when callose was deposited at the fiber base but became evident at the time of callose degradation. Genotypically, the level of GhGluc1 expression was high in the short fiber genotype and weak in the intermediate and long fiber genotypes. The data provide genotypic and developmental evidence that (1) plasmodesmata closure appears to play an important role in elongating cotton fibers, (2) callose deposition and degradation may be involved in the plasmodesmata closure and reopening, respectively, and (3) the expression of GhGluc1 could play a role in this process by degrading callose, thus opening the plasmodesmata.

Anatomical and Transcriptomic Studies of the Coleorhiza Reveal the Importance of This Tissue in Regulating Dormancy in Barley

The decay of seed dormancy during after-ripening is not well understood, but elucidation of the mechanisms involved may be important for developing strategies for modifying dormancy in crop species and, for example, addressing the problem of preharvest sprouting in cereals. We have studied the germination characteristics of barley (Hordeum vulgare ‘Betzes’) embryos, including a description of anatomical changes in the coleorhiza and the enclosed seminal roots. The changes that occur correlate with abscisic acid (ABA) contents of embryo tissues. To understand the molecular mechanisms involved in dormancy loss, we compared the transcriptome of dormant and after-ripened barley embryos using a tissue-specific microarray approach. Our results indicate that in the coleorhiza, ABA catabolism is promoted and ABA sensitivity is reduced and that this is associated with differential regulation by after-ripening of ABA 8′-hydroxylase and of the LIPID PHOSPHATE PHOSPHATASE gene family and ABI3-INTERACTING PROTEIN2, respectively. We also identified other processes, including jasmonate responses, cell wall modification, nitrate and nitrite reduction, mRNA stability, and blue light sensitivity, that were affected by after-ripening in the coleorhiza that may be downstream of ABA signaling. Based on these results, we propose that the coleorhiza plays a major role in causing dormancy by acting as a barrier to root emergence and that after-ripening potentiates molecular changes related to ABA metabolism and sensitivity that ultimately lead to degradation of the coleorhiza, root emergence, and germination.

Rapid and dynamic subcellular reorganization following mechanical stimulation of Arabidopsis epidermal cells mimics responses to fungal and oomycete attack

BackgroundPlant cells respond to the presence of potential fungal or oomycete pathogens by mounting a basal defence response that involves aggregation of cytoplasm, reorganization of cytoskeletal, endomembrane and other cell components and development of cell wall appositions beneath the infection site. This response is induced by non-adapted, avirulent and virulent pathogens alike, and in the majority of cases achieves penetration resistance against the microorganism on the plant surface. To explore the nature of signals that trigger this subcellular response and to determine the timing of its induction, we have monitored the reorganization of GFP-tagged actin, microtubules, endoplasmic reticulum (ER) and peroxisomes in Arabidopsis plants – after touching the epidermal surface with a microneedle.ResultsWithin 3 to 5 minutes of touching the surface of Arabidopsis cotyledon epidermal cells with fine glass or tungsten needles, actin microfilaments, ER and peroxisomes began to accumulate beneath the point of contact with the needle. Formation of a dense patch of actin was followed by focusing of actin cables on the site of contact. Touching the cell surface induced localized depolymerization of microtubules to form a microtubule-depleted zone surrounding a dense patch of GFP-tubulin beneath the needle tip. The concentration of actin, GFP-tubulin, ER and peroxisomes remained focused on the contact site as the needle moved across the cell surface and quickly dispersed when the needle was removed.ConclusionOur results show that plant cells can detect the gentle pressure of a microneedle on the epidermal cell surface and respond by reorganizing subcellular components in a manner similar to that induced during attack by potential fungal or oomycete pathogens. The results of our study indicate that during plant-pathogen interactions, the basal defence response may be induced by the plants perception of the physical force exerted by the pathogen as it attempts to invade the epidermal cell surface.

Loss of Cellulose Synthase-Like F6 Function Affects Mixed-Linkage Glucan Deposition, Cell Wall Mechanical Properties, and Defense Responses in Vegetative Tissues of Rice

Mixed-linkage glucan (MLG) is a cell wall polysaccharide containing a backbone of unbranched (1,3)- and (1,4)-linked β-glucosyl residues. Based on its occurrence in plants and chemical characteristics, MLG has primarily been associated with the regulation of cell wall expansion due to its high and transient accumulation in young, expanding tissues. The Cellulose synthase-like F (CslF) subfamily of glycosyltransferases has previously been implicated in mediating the biosynthesis of this polymer. We confirmed that the rice (Oryza sativa) CslF6 gene mediates the biosynthesis of MLG by overexpressing it in Nicotiana benthamiana. Rice cslf6 knockout mutants show a slight decrease in height and stem diameter but otherwise grew normally during vegetative development. However, cslf6 mutants display a drastic decrease in MLG content (97% reduction in coleoptiles and virtually undetectable in other tissues). Immunodetection with an anti-MLG monoclonal antibody revealed that the coleoptiles and leaves retain trace amounts of MLG only in specific cell types such as sclerenchyma fibers. These results correlate with the absence of endogenous MLG synthase activity in mutant seedlings and 4-week-old sheaths. Mutant cell walls are weaker in mature stems but not seedlings, and more brittle in both stems and seedlings, compared to wild type. Mutants also display lesion mimic phenotypes in leaves, which correlates with enhanced defense-related gene expression and enhanced disease resistance. Taken together, our results underline a weaker role of MLG in cell expansion than previously thought, and highlight a structural role for MLG in nonexpanding, mature stem tissues in rice.

Phases of Infection and Gene Expression of Fusarium graminearum During Crown Rot Disease of Wheat

Fusarium graminearum causes head blight (FHB) and crown rot (CR) diseases in wheat. Compared with FHB, CR symptom development occurs slowly, usually taking 4 to 8 weeks to become visible. To characterize CR development, we used histological and real-time quantitative polymerase chain reaction analyses to assess fungal colonization during a timecourse of infection. Three distinct phases of infection were identified: i) initial spore germination with formation of a superficial hyphal mat at the inoculation point, ii) colonization of the adaxial epidermis of the outer leaf sheath and mycelial growth from the inoculation point to the crown, concomitant with a drop in fungal biomass, and iii) extensive colonization of the internal crown tissue. Fungal gene expression was examined during each phase using Affymetrix GeneChips. In total, 1,839 F. graminearum genes were significantly upregulated, including some known FHB virulence genes (e.g., TRI5 and TRI14), and 2,649 genes were significantly downregulated in planta compared with axenically cultured mycelia. Global comparisons of fungal gene expression with published data for FHB showed significant similarities between early stages of FHB and CR. These results indicate that CR disease development involves distinct phases of colonization, each of which is associated with a different fungal gene expression program.