Guichuan Hou

RDR6 Has a Broad-Spectrum but Temperature-Dependent Antiviral Defense Role in Nicotiana benthamiana

ABSTRACT SDE1/SGS2/RDR6, a putative RNA-dependent RNA polymerase (RdRP) from Arabidopsis thaliana, has previously been found to be indispensable for maintaining the posttranscriptional silencing of transgenes, but it is seemingly redundant for antiviral defense. To elucidate the antiviral role of this RdRP in a different host plant and to evaluate whether plant growth conditions affect its role, we down-regulated expression of the Nicotiana benthamiana homolog, NbRDR6, and examined the plants for altered susceptibility to various viruses at different growth temperatures. The results we describe here clearly show that plants with reduced expression of NbRDR6 were more susceptible to all viruses tested and that this effect was more pronounced at higher growth temperatures. Diminished expression of NbRDR6 also permitted efficient multiplication of tobacco mosaic virus in the shoot apices, leading to serious disruption with microRNA-mediated developmental regulation. Based on these results, we propose that NbRDR6 participates in the antiviral RNA silencing pathway that is stimulated by rising temperatures but suppressed by virus-encoded silencing suppressors. The relative strengths of these two factors, along with other plant defense components, critically influence the outcome of virus infections.

The Highly Similar Arabidopsis Homologs of Trithorax ATX1 and ATX2 Encode Proteins with Divergent Biochemical Functions

Gene duplication followed by functional specialization is a potent force in the evolution of biological diversity. A comparative study of two highly conserved duplicated genes, ARABIDOPSIS TRITHORAX-LIKE PROTEIN1 (ATX1) and ATX2, revealed features of both partial redundancy and of functional divergence. Although structurally similar, their regulatory sequences have diverged, resulting in distinct temporal and spatial patterns of expression of the ATX1 and ATX2 genes. We found that ATX2 methylates only a limited fraction of nucleosomes and that ATX1 and ATX2 influence the expression of largely nonoverlapping gene sets. Even when coregulating shared targets, ATX1 and ATX2 may employ different mechanisms. Most remarkable is the divergence of their biochemical activities: both proteins methylate K4 of histone H3, but while ATX1 trimethylates it, ATX2 dimethylates it. ATX2 and ATX1 provide an example of separated K4 di from K4 trimethyltransferase activity.

Enhanced gravitropism of roots with a disrupted cap actin cytoskeleton

The actin cytoskeleton has been proposed to be a major player in plant gravitropism. However, understanding the role of actin in this process is far from complete. To address this problem, we conducted an analysis of the effect of Latrunculin B (Lat B), a potent actin-disrupting drug, on root gravitropism using various parameters that included detailed curvature kinetics, estimation of gravitropic sensitivity, and monitoring of curvature development after extended clinorotation. Lat B treatment resulted in a promotion of root curvature after a 90° reorientation in three plant species tested. More significantly, the sensitivity of maize (Zea mays) roots to gravity was enhanced after actin disruption, as determined from a comparison of presentation time of Lat B-treated versus untreated roots. A short 10-min gravistimulus followed by extended rotation on a 1-rpm clinostat resulted in extensive gravitropic responses, manifested as curvature that often exceeded 90°. Application of Lat B to the cap or elongation zone of maize roots resulted in the disruption of the actin cytoskeleton, which was confined to the area of localized Lat B application. Only roots with Lat B applied to the cap displayed the strong curvature responses after extended clinorotation. Our study demonstrates that disrupting the actin cytoskeleton in the cap leads to the persistence of a signal established by a previous gravistimulus. Therefore, actin could function in root gravitropism by providing a mechanism to regulate the proliferation of a gravitropic signal originating from the cap to allow the root to attain its correct orientation or set point angle.

Functional Analysis of the Cellulose Synthase-Like Genes CSLD1, CSLD2, and CSLD4 in Tip-Growing Arabidopsis Cells

A reverse genetic approach was used to investigate the functions of three members of the cellulose synthase superfamily in Arabidopsis (Arabidopsis thaliana), CELLULOSE SYNTHASE-LIKE D1 (CSLD1), CSLD2, and CSLD4. CSLD2 is required for normal root hair growth but has a different role from that previously described for CSLD3 (KOJAK). CSLD2 is required during a later stage of hair development than CSLD3, and CSLD2 mutants produce root hairs with a range of abnormalities, with many root hairs rupturing late in development. Remarkably, though, it was often the case that in CSLD2 mutants, tip growth would resume after rupturing of root hairs. In silico, semiquantitative reverse transcription-polymerase chain reaction, and promoter-reporter construct analyses indicated that the expression of both CSLD2 and CSLD3 is elevated at reduced temperatures, and the phenotypes of mutants homozygous for insertions in these genes were partially rescued by reduced temperature growth. However, this was not the case for a double mutant homozygous for insertions in both CSLD2 and CSLD3, suggesting that there may be partial redundancy in the functions of these genes. Mutants in CSLD1 and CSLD4 had a defect in male transmission, and plants heterozygous for insertions in CSLD1 or CSLD4 were defective in their ability to produce pollen tubes, although the number and morphology of pollen grains was normal. We propose that the CSLD family of putative glycosyltransferases synthesize a polysaccharide that has a specialized structural role in the cell walls of tip-growing cells.

Acetylesterase-Mediated Deacetylation of Pectin Impairs Cell Elongation, Pollen Germination, and Plant Reproduction

Some sugar residues of the pectinaceous polysaccharide backbone of primary cell walls are acetylated. This study identifies and characterizes a Populus pectin acetylesterase that liberates acetylesters from polysaccharides and unveils its biological roles in planta as a structural regulator of cell wall extensibility and digestibility that affects plant growth, development, and reproduction. Pectin is a major component of the primary cell wall of higher plants. Some galacturonyl residues in the backbone of pectinaceous polysaccharides are often O-acetylated at the C-2 or C-3 position, and the resulting acetylesters change dynamically during the growth and development of plants. The processes involve both enzymatic acetylation and deacetylation. Through genomic sequence analysis, we identified a pectin acetylesterase (PAE1) from black cottonwood (Populus trichocarpa). Recombinant Pt PAE1 exhibited preferential activity in releasing the acetate moiety from sugar beet (Beta vulgaris) and potato (Solanum tuberosum) pectin in vitro. Overexpressing Pt PAE1 in tobacco (Nicotiana tabacum) decreased the level of acetyl esters of pectin but not of xylan. Deacetylation engendered differential changes in the composition and/or structure of cell wall polysaccharides that subsequently impaired the cellular elongation of floral styles and filaments, the germination of pollen grains, and the growth of pollen tubes. Consequently, plants overexpressing PAE1 exhibited severe male sterility. Furthermore, in contrast to the conventional view, PAE1-mediated deacetylation substantially lowered the digestibility of pectin. Our data suggest that pectin acetylesterase functions as an important structural regulator in planta by modulating the precise status of pectin acetylation to affect the remodeling and physiochemical properties of the cell walls polysaccharides, thereby affecting cell extensibility.

Heteroblastic Root Development in Ceratopteris richardii (Parkeriaceae)

Heteroblastic variation among leaves arising from the shoot system is a well‐known phenomenon, but relatively little effort has been directed at assessing whether comparable variation occurs among shoot‐borne roots in homorhizic plants. A morphological and anatomical study of shoot‐borne root formation in the homosporous fern Ceratopteris richardii revealed two developmentally distinct root populations. Roots in the first population were produced once each shoot plastochron, with a single organ originating just beneath each leaf. Among the first six of these roots produced by young shoots, the root plastochron and individual growth rate, the final organ size (diameter and length), the anatomy, and the onset of lateral root initiation all varied as a function of shoot age. The positional differences observed among these organs constituted the first portion of a root heteroblastic series that continued into the adult shoot. The second root population arose from leaf bases beginning near the eighth node, with roots becoming more numerous on leaves at successive nodes. This secondary form of shoot‐borne rooting behavior constituted another aspect of heteroblastic variation, but it is attributed to differences in developmental potential among leaves along the shoot and not to root heteroblasty in a strict sense. The results underscore basic differences in the morphological relationship between root and shoot systems in homorhizic ferns compared with seed plants and show that C. richardii is a useful model for investigating aspects of phase change related to rooting behavior that are not represented in flowering plants.

Delineation of VEGF-regulated genes and functions in the cervix of pregnant rodents by DNA microarray analysis

BackgroundVEGF-regulated genes in the cervices of pregnant and non-pregnant rodents (rats and mice) were delineated by DNA microarray and Real Time PCR, after locally altering levels of or action of VEGF using VEGF agents, namely siRNA, VEGF receptor antagonist and mouse VEGF recombinant protein.MethodsTissues were analyzed by genome-wide DNA microarray analysis, Real-time and gel-based PCR, and SEM, to decipher VEGF function during cervical remodeling. Data were analyzed by EASE score (microarray) and ANOVA (Real Time PCR) followed by Scheffes F-test for multiple comparisons.ResultsOf the 30,000 genes analyzed, about 4,200 genes were altered in expression by VEGF, i.e., expression of about 2,400 and 1,700 genes were down- and up-regulated, respectively. Based on EASE score, i.e., grouping of genes according to their biological process, cell component and molecular functions, a number of vascular- and non-vascular-related processes were found to be regulated by VEGF in the cervix, including immune response (including inflammatory), cell proliferation, protein kinase activity, and cell adhesion molecule activity. Of interest, mRNA levels of a select group of genes, known to or with potential to influence cervical remodeling were altered. For example, real time PCR analysis showed that levels of VCAM-1, a key molecule in leukocyte recruitment, endothelial adhesion, and subsequent trans-endothelial migration, were elevated about 10 folds by VEGF. Further, VEGF agents also altered mRNA levels of decorin, which is involved in cervical collagen fibrillogenesis, and expression of eNO, PLC and PKC mRNA, critical downstream mediators of VEGF. Of note, we show that VEGF may regulate cervical epithelial proliferation, as revealed by SEM.ConclusionThese data are important in that they shed new insights in VEGFs possible roles and mechanisms in cervical events near-term, including cervical remodeling.

Developmental anatomy of the fifth shoot-borne root in young sporophytes of Ceratopteris richardii

Young sporophytes of the homosporous fern Ceratopteris richardii produce a single shoot-borne root below each leaf. The developmental anatomy of the fifth sporophyte root is described using scanning electron microscopy and histological techniques. Three merophyte orthostichies in the body of the root originate from three proximal division faces of a tetrahedral root apical cell. Eight or nine divisions occur in a relatively regular sequence within each merophyte and produce a characteristic radial anatomical pattern in the root. The exact number of early divisions within a merophyte depends on the merophyte’s position within the root as a whole. Predictable inter-merophyte differences arise because a 2-fold (diarch) anatomical symmetry that is characteristic of mature roots is superimposed on a 3-fold radial symmetry that originates behind the apical cell. Before early formative divisions within a merophyte are completed, additional proliferative divisions begin to increase the number of cells within previously established tissue zones. The cellular parameters of early fifth root development in C. richardii are relatively invariant, and are reminiscent of patterns previously described for the heterosporous fern Azolla. Young sporophytes of C. richardii provide a useful model to further investigate the genetic regulation of root development in a non-seed plant, where the anatomy of meristem organization differs from that seen in flowering plant species.

ABA, ROS and NO are Key Players During Switchgrass Seed Germination

Seed dormancy and germination are complex physiological processes usually under hormonal control. Germination of seeds from many plants including switchgrass, are inhibited by ABA and promoted by NO or ROS. However, ABA apparently requires both ROS and NO as intermediates in its action, with ROS produced by membrane-bound NADPH-oxidases responsive to ABA. In switchgrass seeds, externally supplied hydrogen peroxide (ROS), but not NO will overcome ABA-imposed inhibition of germination. Stimulation of germination by external ROS can be partially blocked by NO-scavengers, suggesting that NO is required for seed germination in switchgrass as well as for ABA-induced inhibition of germination. Collectively, these data suggest that multiple mechanisms might be required to sense and respond to varying levels of ABA, NO and ROS in switchgrass seeds.

The MYB107 Transcription Factor Positively Regulates Suberin Biosynthesis

The Arabidopsis MYB107 transcription factor regulates the expression of suberin biosynthetic genes in developing seeds, thereby controlling the synthesis and assembly of seed coat suberin. Suberin, a lipophilic polymer deposited in the outer integument of the Arabidopsis (Arabidopsis thaliana) seed coat, represents an essential sealing component controlling water and solute movement and protecting seed from pathogenic infection. Although many genes responsible for suberin synthesis are identified, the regulatory components controlling its biosynthesis have not been definitively determined. Here, we show that the Arabidopsis MYB107 transcription factor acts as a positive regulator controlling suberin biosynthetic gene expression in the seed coat. MYB107 coexpresses with suberin biosynthetic genes in a temporal manner during seed development. Disrupting MYB107 particularly suppresses the expression of genes involved in suberin but not cutin biosynthesis, lowers seed coat suberin accumulation, alters suberin lamellar structure, and consequently renders higher seed coat permeability and susceptibility to abiotic stresses. Furthermore, MYB107 directly binds to the promoters of suberin biosynthetic genes, verifying its primary role in regulating their expression. Identifying MYB107 as a positive regulator for seed coat suberin synthesis offers a basis for discovering the potential transcriptional network behind one of the most abundant lipid-based polymers in nature.