Over-expression of AtEXLA2 alters etiolated arabidopsis hypocotyl growth

Background and Aims Plant stature and shape are largely determined by cell elongation, a process that is strongly controlled at the level of the cell wall. This is associated with the presence of many cell wall proteins implicated in the elongation process. Several proteins and enzyme families have been suggested to be involved in the controlled weakening of the cell wall, and these include xyloglucan endotransglucosylases/hydrolases (XTHs), yieldins, lipid transfer proteins and expansins. Although expansins have been the subject of much research, the role and involvement of expansin-like genes/proteins remain mostly unclear. This study investigates the expression and function of AtEXLA2 (At4g38400), a member of the expansin-like A (EXLA) family in arabidposis, and considers its possible role in cell wall metabolism and growth.Methods Transgenic plants of Arabidopsis thaliana were grown, and lines over-expressing AtEXLA2 were identified. Plants were grown in the dark, on media containing growth hormones or precursors, or were gravistimulated. Hypocotyls were studied using transmission electron microscopy and extensiometry. Histochemical GUS (β-glucuronidase) stainings were performed.Key Results AtEXLA2 is one of the three EXLA members in arabidopsis. The protein lacks the typical domain responsible for expansin activity, but contains a presumed cellulose-interacting domain. Using promoter::GUS lines, the expression of AtEXLA2 was seen in germinating seedlings, hypocotyls, lateral root cap cells, columella cells and the central cylinder basally to the elongation zone of the root, and during different stages of lateral root development. Furthermore, promoter activity was detected in petioles, veins of leaves and filaments, and also in the peduncle of the flowers and in a zone just beneath the papillae. Over-expression of AtEXLA2 resulted in an increase of >10 % in the length of dark-grown hypocotyls and in slightly thicker walls in non-rapidly elongating etiolated hypocotyl cells. Biomechanical analysis by creep tests showed that AtEXLA2 over-expression may decrease the wall strength in arabidopsis hypocotyls.Conclusions It is concluded that AtEXLA2 may function as a positive regulator of cell elongation in the dark-grown hypocotyl of arabidopsis by possible interference with cellulose metabolism, deposition or its organization.     

AGO1 controls arabidopsis inflorescence architecture possibly by regulating TFL1 expression

Background and Aims

The TERMINAL FLOWER 1 (TFL1) gene is pivotal in the control of inflorescence architecture in arabidopsis. Thus, tfl1 mutants flower early and have a very short inflorescence phase, while TFL1-overexpressing plants have extended vegetative and inflorescence phases, producing many coflorescences. TFL1 is expressed in the shoot meristems, never in the flowers. In the inflorescence apex, TFL1 keeps the floral genes LEAFY (LFY) and APETALA1 (AP1) restricted to the flower, while LFY and AP1 restrict TFL1 to the inflorescence meristem. In spite of the central role of TFL1 in inflorescence architecture, regulation of its expression is poorly understood. This study aims to expand the understanding of inflorescence development by identifying and studying novel TFL1 regulators.

Methods

Mutagenesis of an Arabidopsis thaliana line carrying a TFL1::GUS (β-glucuronidase) reporter construct was used to isolate a mutant with altered TFL1 expression. The mutated gene was identified by positional cloning. Expression of TFL1 and TFL1::GUS was analysed by real-time PCR and histochemical GUS detection. Double-mutant analysis was used to assess the contribution of TFL1 to the inflorescence mutant phenotype.

Key Results

A mutant with both an increased number of coflorescences and high and ectopic TFL1 expression was isolated. Cloning of the mutated gene showed that both phenotypes were caused by a mutation in the ARGONAUTE1 (AGO1) gene, which encodes a key component of the RNA silencing machinery. Analysis of another ago1 allele indicated that the proliferation of coflorescences and ectopic TFL1 expression phenotypes are not allele specific. The increased number of coflorescences is suppressed in ago1 tfl1 double mutants.

Conclusions

The results identify AGO1 as a repressor of TFL1 expression. Moreover, they reveal a novel role for AGO1 in inflorescence development, controlling the production of coflorescences. AGO1 seems to play this role through regulating TFL1 expression.     

ZEITLUPE positively regulates hypocotyl elongation at warm temperature under light in Arabidopsis thaliana

Hypocotyl cell elongation has been studied as a model to understand how cellular expansion contributes to plant organ growth. Hypocotyl elongation is affected by multiple environmental factors, including light quantity and light quality. Red light inhibits hypocotyl growth via the phytochrome signaling pathways. Proteins of the FLAVIN-BINDING KELCH REPEAT F-BOX 1 / LOV KELCH PROTEIN 2 / ZEITLUPE family are positive regulators of hypocotyl elongation under red light in Arabidopsis. These proteins were suggested to reduce phytochrome-mediated inhibition of hypocotyl elongation. Here, we show that ZEITLUPE also functions as a positive regulator in warmth-induced hypocotyl elongation under light in Arabidopsis.     

Gene expression profile of zeitlupe/lov kelch protein1 T-DNA insertion mutants in Arabidopsis thaliana: Downregulation of auxin-inducible genes in hypocotyls

Elongation of hypocotyl cells has been studied as a model for elucidating the contribution of cellular expansion to plant organ growth. ZEITLUPE (ZTL) or LOV KELCH PROTEIN1 (LKP1) is a positive regulator of warmth-induced hypocotyl elongation under white light in Arabidopsis, although the molecular mechanisms by which it promotes hypocotyl cell elongation remain unknown. Microarray analysis showed that 134 genes were upregulated and 204 genes including 15 auxin-inducible genes were downregulated in the seedlings of 2 ztl T-DNA insertion mutants grown under warm conditions with continuous white light. Application of a polar auxin transport inhibitor, an auxin antagonist or an auxin biosynthesis inhibitor inhibited hypocotyl elongation of control seedlings to the level observed with the ztl mutant. Our data suggest the involvement of auxin and auxin-inducible genes in ZTL-mediated hypocotyl elongation.     

Molecular control of seasonal flowering in rice,arabidopsis and temperate cereals

Background

Rice (Oryza sativa) and Arabidopsis thaliana have been widely used as model systems to understand how plants control flowering time in response to photoperiod and cold exposure. Extensive research has resulted in the isolation of several regulatory genes involved in flowering and for them to be organized into a molecular network responsive to environmental cues. When plants are exposed to favourable conditions, the network activates expression of florigenic proteins that are transported to the shoot apical meristem where they drive developmental reprogramming of a population of meristematic cells. Several regulatory factors are evolutionarily conserved between rice and arabidopsis. However, other pathways have evolved independently and confer specific characteristics to flowering responses.

Scope

This review summarizes recent knowledge on the molecular mechanisms regulating daylength perception and flowering time control in arabidopsis and rice. Similarities and differences are discussed between the regulatory networks of the two species and they are compared with the regulatory networks of temperate cereals, which are evolutionarily more similar to rice but have evolved in regions where exposure to low temperatures is crucial to confer competence to flower. Finally, the role of flowering time genes in expansion of rice cultivation to Northern latitudes is discussed.

Conclusions

Understanding the mechanisms involved in photoperiodic flowering and comparing the regulatory networks of dicots and monocots has revealed how plants respond to environmental cues and adapt to seasonal changes. The molecular architecture of such regulation shows striking similarities across diverse species. However, integration of specific pathways on a basal scheme is essential for adaptation to different environments. Artificial manipulation of flowering time by means of natural genetic resources is essential for expanding the cultivation of cereals across different environments.     

Cycling of clock genes entrained to the solar rhythm enables plants to tell time: data from arabidopsis

Background and Aims An endogenous rhythm synchronized to dawn cannot time photosynthesis-linked genes to peak consistently at noon since the interval between sunrise and noon changes seasonally. In this study, a solar clock model that circumvents this limitation is proposed using two daily timing references synchronized to noon and midnight. Other rhythmic genes that are not directly linked to photosynthesis, and which peak at other times, also find an adaptive advantage in entrainment to the solar rhythm.Methods Fourteen datasets extracted from three published papers were used in a meta-analysis to examine the cyclic behaviour of the Arabidopsis thaliana photosynthesis-related gene CAB2 and the clock oscillator genes TOC1 and LHY in T cycles and N–H cycles.Key Results Changes in the rhythms of CAB2, TOC1 and LHY in plants subjected to non-24-h light:dark cycles matched the hypothesized changes in their behaviour as predicted by the solar clock model, thus validating it. The analysis further showed that TOC1 expression peaked ∼5·5 h after mid-day, CAB2 peaked close to noon, while LHY peaked ∼7·5 h after midnight, regardless of the cycle period, the photoperiod or the light:dark period ratio. The solar clock model correctly predicted the zeitgeber timing of these genes under 11 different lighting regimes comprising combinations of seven light periods, nine dark periods, four cycle periods and four light:dark period ratios. In short cycles that terminated before LHY could be expressed, the solar clock correctly predicted zeitgeber timing of its expression in the following cycle.Conclusions Regulation of gene phases by the solar clock enables the plant to tell the time, by which means a large number of genes are regulated. This facilitates the initiation of gene expression even before the arrival of sunrise, sunset or noon, thus allowing the plant to ‘anticipate’ dawn, dusk or mid-day respectively, independently of the photoperiod.     

Diurnal changes in shoot water dynamics are synchronized with hypocotyl elongation in Arabidopsis thaliana

We recently demonstrated the circadian clock modulated water dynamics in the roots of a small model plant, Arabidopsis thaliana, by the Nuclear Magnetic Resonance (NMR) microimaging technique. Our developed technique was able to visualize the water distribution that depended on differences in the ¹H signal among region in the shoot, such as the shoot apex, the hypocotyl and the root shoot junction. Water content in the shoot increased during periods of light in comparison with dark periods, and continued through the early stage of seedling growth until the dark period. When the water content changed, elongation and/or movement occurred in the hypocotyl, and these events were synchronized. The water dynamics of the shoot also displayed an opposite phase with the root water dynamics.     

MIZ1-regulated hydrotropism functions in the growth and survival of Arabidopsis thaliana under natural conditions

Background and Aims

Root hydrotropism is a response to water-potential gradients that makes roots bend towards areas of higher water potential. The gene MIZU-KUSSEI1 (MIZ1) that is essential for hydrotropism in Arabidopsis roots has previously been identified. However, the role of root hydrotropism in plant growth and survival under natural conditions has not yet been proven. This study assessed how hydrotropic response contributes to drought avoidance in nature.

Methods

An experimental system was established for the study of Arabidopsis hydrotropism in soil. Characteristics of hydrotropism were analysed by comparing the responses of the miz1 mutant, transgenic plants overexpressing MIZ1 (MIZ1OE) and wild-type plants.

Key Results

Wild-type plants developed root systems in regions with higher water potential, whereas the roots of miz1 mutant plants did not show a similar response. This pattern of root distribution induced by hydrotropism was more pronounced in MIZ1OE plants than in wild-type plants. In addition, shoot biomass and the number of plants that survived under drought conditions were much greater in MIZ1OE plants.

Conclusions

These results show that hydrotropism plays an important role in root system development in soil and contributes to drought avoidance, which results in a greater yield and plant survival under water-limited conditions. The results also show that MIZ1 overexpression can be used for improving plant productivity in arid areas.     

High frequency of plant regeneration from hypocotyl explants of Brassica carinata A. Br.

An efficient tissue culture system for high frequency of plant regeneration from hypocotyl explants of Brassica carinata was developed via manipulation of culture medium and selection of explants. Explants grown on medium containing combinations of 2 mg l^-1 BA and 0.01 mg l^-1 NAA or 4 mg l^-1 kinetin and 0.01 mg l^-1 2,4-D regenerated shoots at 100% frequency. High frequency shoot regeneration occurred only from explants originating from 6 to 7-day-old but not younger or older seedlings. Explants showed higher regeneration capacity at the distal end than the proximal end, and the upper segment was more regenerative than the lower segment of hypocotyl. Regenerants were rooted on half-strength growth regulator-free medium, acclimatized and developed into normal, fertile plants.Abbreviations BA benzyladenine - 2-4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - MS Murashige & Skoog     

Nitric oxide is required for the auxin-induced activation of NADPH-dependent thioredoxin reductase and protein denitrosylation during root growth responses in arabidopsis

Background and Aims Auxin is the main phytohormone controlling root development in plants. This study uses pharmacological and genetic approaches to examine the role of auxin and nitric oxide (NO) in the activation of NADPH-dependent thioredoxin reductase (NTR), and the effect that this activity has on root growth responses in Arabidopsis thaliana.Methods Arabidopsis seedlings were treated with auxin with or without the NTR inhibitors auranofin (ANF) and 1-chloro-2, 4-dinitrobenzene (DNCB). NTR activity, lateral root (LR) formation and S-nitrosothiol content were measured in roots. Protein S-nitrosylation was analysed by the biotin switch method in wild-type arabidopsis and in the double mutant ntra ntrb.Key Results The auxin-mediated induction of NTR activity is inhibited by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO), suggesting that NO is downstream of auxin in this regulatory pathway. The NTR inhibitors ANF and DNCB prevent auxin-mediated activation of NTR and LR formation. Moreover, ANF and DNCB also inhibit auxin-induced DR5 : : GUS and BA3 : : GUS gene expression, suggesting that the auxin signalling pathway is compromised without full NTR activity. Treatment of roots with ANF and DNCB increases total nitrosothiols (SNO) content and protein S-nitrosylation, suggesting a role of the NTR-thioredoxin (Trx)-redox system in protein denitrosylation. In agreement with these results, the level of S-nitrosylated proteins is increased in the arabidopsis double mutant ntra ntrb as compared with the wild-type.Conclusions The results support for the idea that NTR is involved in protein denitrosylation during auxin-mediated root development. The fact that a high NO concentration induces NTR activity suggests that a feedback mechanism to control massive and unregulated protein S-nitrosylation could be operating in plant cells.     

In Vitro Repression of Brca1-associated RING Domain Gene,Bard1, Induces Phenotypic Changes in Mammary Epithelial Cells

BRCA1-associated RING domain (BARD1) was identified as a protein interacting with the breast cancer gene product BRCA1. The identification of tumorigenic missense mutations within BRCA1 that impair the formation of BARD1–BRCA1 complexes, and of BARD1 mutations in breast carcinomas, sustain the view that BARD1 is involved in BRCA1-mediated tumor suppression. We have cloned the murine Bard1 gene and determined that its expression in different tissues correlates with the expression profile of Brca1. To investigate the function of Bard1, we have reduced Bard1 gene expression in TAC-2 cells, a murine mammary epithelial cell line that retains morphogenetic properties characteristic of normal breast epithelium. Partial repression of Bard1, achieved by the transfection of TAC-2 cells with plasmids constitutively expressing ribozymes or antisense RNAs, resulted in marked phenotypic changes, consisting of altered cell shape, increased cell size, high frequency of multinucleated cells, and aberrant cell cycle progression. Furthermore, Bard1-repressed cell clones overcame contact inhibition of cell proliferation when grown in monolayer cultures and lost the capacity to form luminal structures in three-dimensional collagen gels. These results demonstrate that Bard1 repression induces complex changes in mammary epithelial cell properties which are suggestive of a premalignant phenotype.     

Auxin and cytokinin control formation of the quiescent centre in the adventitious root apex of arabidopsis

Background and Aims

Adventitious roots (ARs) are part of the root system in numerous plants, and are required for successful micropropagation. In the Arabidopsis thaliana primary root (PR) and lateral roots (LRs), the quiescent centre (QC) in the stem cell niche of the meristem controls apical growth with the involvement of auxin and cytokinin. In arabidopsis, ARs emerge in planta from the hypocotyl pericycle, and from different tissues in in vitro cultured explants, e.g. from the stem endodermis in thin cell layer (TCL) explants. The aim of this study was to investigate the establishment and maintenance of the QC in arabidopsis ARs, in planta and in TCL explants, because information about this process is still lacking, and it has potential use for biotechnological applications.

Methods

Expression of PR/LR QC markers and auxin influx (LAX3)/efflux (PIN1) genes was investigated in the presence/absence of exogenous auxin and cytokinin. Auxin was monitored by the DR5::GUS system and cytokinin by immunolocalization. The expression of the auxin-biosynthetic YUCCA6 gene was also investigated by in situ hybridization in planta and in AR-forming TCLs from the indole acetic acid (IAA)-overproducing superroot2-1 mutant and its wild type.

Key Results

The accumulation of auxin and the expression of the QC marker WOX5 characterized the early derivatives of the AR founder cells, in planta and in in vitro cultured TCLs. By determination of PIN1 auxin efflux carrier and LAX3 auxin influx carrier activities, an auxin maximum was determined to occur at the AR tip, to which WOX5 expression was restricted, establishing the positioning of the QC. Cytokinin caused a restriction of LAX3 and PIN1 expression domains, and concomitantly the auxin biosynthesis YUCCA6 gene was expressed in the apex.

Conclusions

In ARs formed in planta and TCLs, the QC is established in a similar way, and auxin transport and biosynthesis are involved through cytokinin tuning.     

Arabidopsis thaliana: Proliferating cell nuclear antigen 1 and 2 possibly form homo- and hetero-trimeric complexes in the plant cell

The proliferating cell nuclear antigen (PCNA) is a key component of the eukaryotic DNA replication machinery. It also plays an important role in DNA repair mechanisms. Despite the intense scientific research on yeast and human PCNA, information describing the function of this protein in plants is still very limited. In the previous study Arabidopsis PCNA2 but not PCNA1 was proposed to be functionally important in DNA polymerase η-dependent postreplication repair. In addition to the above study, PCNA2 but not PCNA1 was also shown to be necessary for Arabidopsis DNA polymerase λ-dependent oxidative DNA damage bypass. Taking into account the reported differences between PCNA1 and PCNA2, we tested the idea of a possible cooperation between PCNA1 and PCNA2 in the plant cell. In a bimolecular fluorescence complementation assay an interaction between PCNA1 and PCNA2 was observed in the nucleus, as well as in the cytoplasm. This finding, together with our previous results, indicates that PCNA1 and PCNA2 may cooperate in planta by forming homo- and heterotrimeric rings. The observed interaction might be relevant when distinct functions for PCNA1 and PCNA2 are considered.     

Spatiotemporal variation of leaf epidermal cell growth: a quantitative analysis of Arabidopsis thaliana wild-type and triple cyclinD3 mutant plants

Background and Aims

The epidermis of an expanding dicot leaf is a mosaic of cells differing in identity, size and differentiation stage. Here hypotheses are tested that in such a cell mosaic growth is heterogeneous and changes with time, and that this heterogeneity is not dependent on the cell cycle regulation per se.

Methods

Shape, size and growth of individual cells were followed with the aid of sequential replicas in expanding leaves of wild-type Arabidopsis thaliana and triple cyclinD3 mutant plants, and combined with ploidy estimation using epi-fluorescence microscopy.

Key Results

Relative growth rates in area of individual epidermal cells or small cell groups differ several fold from those of adjacent cells, and change in time. This spatial and temporal variation is not related to the size of either the cell or the nucleus. Shape changes and growth within an individual cell are also heterogeneous: anticlinal wall waviness appears at different times in different wall portions; portions of the cell periphery in contact with different neighbours grow with different rates. This variation is not related to cell growth anisotropy. The heterogeneity is typical for both the wild type and cycD3.

Conclusions

Growth of leaf epidermis exhibits spatiotemporal variability.