SciCombinator

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Journal: The Plant journal : for cell and molecular biology

28

The pattern of cell division, growth and separation during leaf development determines the pattern and amount of airspace in a leaf. The resulting balance of cellular material and airspace is expected to significantly influence the primary function of the leaf, photosynthesis, yet the manner and degree to which cell division patterns impact airspace networks and photosynthesis remains largely unexplored. In this paper we investigate the relationship of cell size and patterning, airspace and photosynthesis by promoting and repressing the expression of cell cycle genes in the leaf mesophyll. Using microCT imaging to quantify leaf cellular architecture and fluorescence/gas exchange analysis to measure leaf function, we show that increased cell density in the mesophyll of Arabidopsis can be used to increase leaf photosynthetic capacity. Our analysis suggests this occurs both by increasing tissue density (decreasing the relative amount of airspace) and by altering the pattern of airspace distribution within the leaf. Our results indicate that cell division patterns influence the photosynthetic performance of a leaf and that it is possible to engineer improved photosynthesis via this approach. This article is protected by copyright. All rights reserved.

Concepts: Chromosome, Bacteria, Cell nucleus, Organelle, Cell division, Cell, Eukaryote, Leaf

28

A eukaryotic chromosome consists of a centromere, two telomeres and a number of replication origins, and “artificial chromosomes” can be created in yeast and mammals when these three elements are artificially joined and introduced into cells. Plant artificial chromosomes (PACs) have been anticipated as new vectors for the development of new crops and as tools for basic research on chromosomes. However, indisputable PAC formation has not yet been confirmed. Here, we present a method for generating PACs in the model plant Arabidopsis thaliana using the Cre/LoxP and Activator (Ac)/Dissociation (Ds) element systems. The successfully generated PAC, designated “AtARC1 (A. thaliana artificial ring chromosome 1)”, originated from a centromeric edge of the long arm of chromosome 2, yet the size (2.85 Mb) is much smaller than that of the original chromosome (26.3 Mb). Although AtARC1 contains only a short centromere domain consisting of 180-bp repeats approximately 250 kb in length, compared with the domain on the original chromosome 2 (3 Mb), the centromere-specific histone H3 (HTR12) was detected on the centromeric region. This result supported the observed stability of the PAC during mitosis in the absence of selection and the transmission of the PAC to the next generation through meiosis. Because AtARC1 contains a unique LoxP site driven by the CaMV 35S promoter, it is possible to introduce a selectable marker and desired transgenes into AtARC1 at the LoxP site using the Cre recombinase. Therefore, AtARC1 meets the criteria for a PAC and is a promising vector. © 2013 The Authors. The Plant Journal © 2013 Blackwell Publishing Ltd.

Concepts: Meiosis, Model organism, Histone, Chromosomes, Eukaryote, Arabidopsis thaliana, DNA, Chromosome

27

The pathogen Pseudomonas syringae requires a type III protein secretion system and the effector proteins it injects into plant cells for pathogenesis. The primary role for P. syringae type III effectors is the suppression of plant immunity. The P. syringae pv. tomato DC3000 HopK1 type III effector was known to suppress the hypersensitive response (HR), a programmed cell death response associated with effector-triggered immunity. Here we show that DC3000 hopK1 mutants are reduced in their ability to grow in Arabidopsis and produce reduced disease symptoms. Arabidopsis transgenically expressing HopK1 are reduced in PAMP-triggered immune responses compared to wild type plants. An N-terminal region of HopK1 shares similarity with the corresponding region in the well-studied type III effector AvrRps4, however, their C-terminal regions are dissimilar indicating that they have different effector activities. HopK1 is processed in planta at the same processing site found in AvrRps4. The processed forms of HopK1 and AvrRps4 are chloroplast-localized indicating that the shared N-terminal regions of these type III effectors represent a chloroplast transit peptide. HopK1’s virulence contribution and the ability of HopK1 and AvrRps4 to suppress immunity required their respective transit peptides, but the AvrRps4-induced HR did not. Our results suggest that a primary virulence target of these type III effectors resides in chloroplasts and that the recognition of AvrRps4 by the plant immune system occurs elsewhere. Moreover, our results reveal that distinct type III effectors utilize a cleavable transit peptide to localize to chloroplasts and that targets within this organelle are important for immunity. This article is protected by copyright. All rights reserved.

Concepts: Cell, Pseudomonas syringae, Innate immune system, Chloroplast, Eukaryote, Protein, Photosynthesis, Immune system

27

We have suggested that, in Arabidopsis, auxin controls the timing of anther dehiscence, possibly by preventing premature endothecium lignification. We show here that auxin content in anthers peaks before the beginning of dehiscence and declines when endothecium lignification occurs. We show that in the auxin-perception mutants afb1-3 and tir1afb2afb3 endothecium lignification and anther dehiscence occur earlier and the gene encoding the transcription factor MYB26, required for endothecium lignification, is overexpressed specifically at early stages; in agreement, MYB26 expression is reduced in NAA-treated anthers, and afb1myb26 double mutants show no endothecial lignification, suggesting that auxin acts through MYB26. As jasmonic acid (JA) controls anther dehiscence, we analysed how auxin and JA interact. In the JA-defective opr3 mutant, indehiscent anthers have a regular timing of endothecium lignification suggesting that JA does not control this event. We show that expression of OPR3 and DAD1 JA biosynthetic genes is enhanced in afb1-3 and tir1afb2afb3 flower buds, whereas it is reduced in NAA-treated flower buds, suggesting that auxin negatively regulates JA biosynthesis. The double mutant afb1opr3 shows premature endothecium lignification, like in afb1-3, and indehiscent anthers due to lack of JA, which is required for stomium opening. By treating afb1opr3 and opr3 inflorescences with JA we show that a high JA content, and precocious endothecium lignification both contribute in inducing early anther dehiscence. We propose that auxin controls anther dehiscence timing by negatively regulating two key events: endothecium lignification via MYB26, and stomium opening via the control of JA biosynthesis. © 2013 The Authors. The Plant Journal © 2013 Blackwell Publishing Ltd.

Concepts: Flower, Silique, Dehiscence, Plant physiology, Plant morphology

27

We have identified in apple (Malus x domestica) three chalcone synthase (CHS) genes. In order to understand the functional redundancy of this gene family RNAi, knockout lines were generated where all three of these genes were down-regulated. These lines had no detectable anthocyanins and radically reduced concentrations of dihydrochalcones and flavonoids. Surprisingly, down-regulation of CHS also led to major changes in plant development, resulting in plants with shortened internode lengths, smaller leaves and a greatly reduced growth rate. Microscopic analysis revealed that these phenotypic changes extended down to the cellular level, with CHS-silenced lines showing aberrant cellular organisation in the leaves. Fruit collected from one CHS-silenced line were smaller than the ‘Royal Gala’ controls, lacked flavonoids in the skin and flesh and also had changes in cell morphology. Auxin transport experiments showed increased altered rates of auxin transport in a CHS-silenced line compared with the ‘Royal Gala control. As flavonoids are well known to be key modulators of auxin transport, we hypothesise that the removal of almost all flavonoids from the plant by CHS silencing creates a vastly altered environment for auxin transport to occur and results in the observed changes in growth and development. © 2013 The New Zealand Institute for Plant & Food Research Ltd The Plant Journal © 2013 Blackwell Publishing Ltd.

Concepts: Virus, Chalcone synthase, Wiley-Blackwell, Fruit, Plant morphology, RNA interference, RNA, Gene

25

Photomorphogenesis is an essential program in plant development. This process is affected by the balanced cooperation of many factors under light and dark conditions. In a previous study, we showed that MYBH is involved in cell elongation. To expand our understanding of MYBH function, we performed a yeast two-hybrid assay and identified a MYB-like Domain (MYBD) transcription factor. In this study, we investigated the function of MYBD, which is a MYBH homolog involved in anthocyanin accumulation. MYBD expression increased in response to light or cytokinin, and MYBD enhanced anthocyanin biosynthesis via the repression of MYBL2 encoding for a transcription factor that had a negative effect on this process. In addition, MYBD can bind in vivo to the MYBL2 promoter and a lower level of histone H3K9 acetylation (H3K9ac) at upstream region of MYBL2 in MYBD-OX in comparison to wild-type plants, implies that MYBD represses MYBL2 expression via an epigenetic mechanism. HY5 directly binds to the MYBD promoter, which indicates that MYBD acts on HY5 downstream in light or cytokinin triggered signaling pathways and this then leads to anthocyanin accumulation. Our results imply that although MYBD and MYBH are homologs, they act in opposite ways during plant photomorphogenesis and these functions should be examined in further studies. This article is protected by copyright. All rights reserved.

Concepts: Copyright, Two-hybrid screening, RNA polymerase, Transcription factor, Transcription, Gene expression, Gene, DNA

25

Reactive oxygen species (ROS) are produced by and have the potential to be damaging to all aerobic organisms. In photosynthetic organisms, they are an unavoidable byproduct of electron transfer in both the chloroplast and mitochondrion. We employ the reference unicellular green alga, Chlamydomonas reinhardtii, to identify the effect of H2 O2 on gene expression by monitoring the transcriptome changes in a timecourse experiment. Comparison of transcriptomes from cells sampled immediately prior to addition of H2 O2 , and 0.5 and 1 h subsequently revealed 1278 differentially abundant transcripts. Of those transcripts that increase in abundance, many encode proteins involved in ROS detoxification, protein degradation and stress-responses, whereas among those that decrease are transcripts encoding proteins involved in photosynthesis and central carbon metabolism. In addition to these transcriptomic adjustments, we observe that H2 O2 addition is followed by an accumulation and oxidation of the total intracellular glutathione pool, and a decrease in photosynthetic O2 output. Additionally, we analyze our transcriptomes in the context of transcript abundance changes in response to singlet O2 (O2 *), and relate our H2 O2 -induced transcripts to a diurnal transcriptome, where we demonstrate enrichments of H2 O2 -induced transcripts early in the light phase, late in the light phase and 2 h prior to light. On this basis several genes that are highlighted in this work may be involved in previously undiscovered stress remediation pathways or acclimation responses. This article is protected by copyright. All rights reserved.

Concepts: Cellular respiration, Reactive oxygen species, Bacteria, DNA, Gene, Photosynthesis, Metabolism, Oxygen

25

Intercellular signaling through trafficking of regulatory proteins is a widespread phenomenon in plants and can deliver positional information for cell fate determination. In the Arabidopsis root meristem, the cell fate determinant SHORT-ROOT (SHR), a GRAS domain transcription factor, acts as a signaling molecule from the stele to the adjacent layer to specify endodermal cell fate. Upon exiting the stele, SHR activates another GRAS domain transcription factor SCARCROW (SCR) which, together with several BIRD/INDETERMINATE DOMAIN proteins, restricts SHR movement to define a single cell layer of endodermis. Here we report that endodermal cell fate also requires the joint activity of both SCR and its closest homologue SCARECROW-LIKE23 (SCL23). We show that SCL23 protein moves with zonation-dependent directionality. Within the meristem, SCL23 exhibits short-ranged ground-tissue-to-vasculature movement. Away from the meristem, SCL23 displays long-range rootward movement into meristematic vasculature and a bidirectional radial spread, respectively. As a known SHR and SCR target, SCL23 also interacts with SCR and SHR and can restrict SHR intercellular outspread without relying on nuclear retention as SCR does. Collectively, our data show that SCL23 is a mobile protein that controls SHR movement and acts redundantly with SCR to specify endodermal fate in the root meristem. This article is protected by copyright. All rights reserved.

Concepts: Meristem, Gene, Bacteria, Signal transduction, Protein, Cell biology, Cell nucleus, DNA

25

The evolutionarily conserved Elongator complex functions in diverse biological processes including salicylic acid-mediated immune response. However, how Elongator functions in jasmonic acid (JA)/ethylene (ET)-mediated defense is unknown. Here, we show that Elongator is required for full induction of the JA/ET defense pathway marker gene PLANT DEFENSIN1.2 (PDF1.2) and for resistance to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola. A loss-of-function mutation in the Arabidopsis Elongator subunit 2 (ELP2) alters B. cinerea-induced transcriptome reprogramming. Interestingly, in elp2, expression of WRKY33, OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59 (ORA59), and PDF1.2 is inhibited, whereas transcription of MYC2 and its target genes is enhanced. However, overexpression of WRKY33 or ORA59 and mutation of MYC2 fail to restore PDF1.2 expression and B. cinerea resistance in elp2, suggesting that ELP2 is required for induction of not only WRKY33 and ORA59 but also PDF1.2. Moreover, elp2 is as susceptible as coronatine-insensitive1 (coi1) and ethylene-insensitive2 (ein2) to B. cinerea, indicating that ELP2 is an important player in B. cinerea resistance. Further analysis of the lesion sizes on the double mutants elp2 coi1 and elp2 ein2 and the corresponding single mutants revealed that the function of ELP2 overlaps with COI1 and is additive to EIN2 for B. cinerea resistance. Finally, basal histone acetylation levels in the coding regions of WRKY33, ORA59, and PDF1.2 are reduced in elp2 and a functional ELP2-GFP fusion protein binds to the chromatin of these genes, suggesting that constitutive ELP2-mediated histone acetylation may be required for full activation of the WRKY33/ORA59/PDF1.2 transcriptional cascade. This article is protected by copyright. All rights reserved.

Concepts: Bacteria, Histone, Evolution, Immune system, Genetics, Gene, Gene expression, DNA

24

HsfB1 is a central regulator of heat stress response and functions dually as transcriptional co-activator of HsfA1a and as general repressor in tomato. HsfB1 is efficiently synthesized during onset of heat stress and rapidly removed in course of attenuation during recovery phase. Initial results point to a complex regime modulating HsfB1 abundance involving the molecular chaperone Hsp90. However, the molecular determinants affecting HsfB1 stability needed to be established. We provide experimental evidence that DNA-bound HsfB1 is efficiently targeted for degradation when active as transcriptional repressor. Manipulation of the DNA-binding affinity by mutating the HsfB1 DNA-binding domain directly influences the stability of the transcription factor. During heat stress HsfB1 is stabilized, probably due to co-activator complex formation with HsfA1a. The process of HsfB1 degradation involves nuclear localized Hsp90. The molecular determinants of HsfB1 turnover identified in here are so far seemingly unique. A mutational switch of the R/KLFGV repressor motif’s arginine and lysine implies that the abundance of other R/KLFGV type Hsfs, if not other transcription factors as well, might be modulated by a comparable mechanism. Thus, we propose a versatile mechanism for strict abundance control of the stress-induced transcription factor HsfB1 for the recovery phase and this mechanism constitutes a form of transcription factor removal from promoters by degradation inside the nucleus. This article is protected by copyright. All rights reserved.

Concepts: Activator, Protein, Promoter, RNA polymerase, Transcription, DNA, Gene expression, Transcription factor