Hyper-activation of the TCP4 transcription factor in Arabidopsis thaliana accelerates multiple aspects of plant maturation

Plant organs are initiated as primordial outgrowths, and require controlled cell division and differentiation to achieve their final size and shape. Superimposed on this is another developmental program that orchestrates the switch from vegetative to reproductive to senescence stages in the life cycle. These require sequential function of heterochronic regulators. Little is known regarding the coordination between organ and organismal growth in plants. The TCP gene family encodes transcription factors that control diverse developmental traits, and a subgroup of class II TCP genes regulate leaf morphogenesis. Absence of these genes results in large, crinkly leaves due to excess division, mainly at margins. It has been suggested that these class II TCPs modulate the spatio-temporal control of differentiation in a growing leaf, rather than regulating cell proliferation per se. However, the link between class II TCP action and cell growth has not been established. As loss-of-function mutants of individual TCP genes in Arabidopsis are not very informative due to gene redundancy, we generated a transgenic line that expressed a hyper-activated form of TCP4 in its endogenous expression domain. This resulted in premature onset of maturation and decreased cell proliferation, leading to much smaller leaves, with cup-shaped lamina in extreme cases. Further, the transgenic line initiated leaves faster than wild-type and underwent precocious reproductive maturation due to a shortened adult vegetative phase. Early senescence and severe fertility defects were also observed. Thus, hyper-activation of TCP4 revealed its role in determining the timing of crucial developmental events, both at the organ and organism level.

The tarani mutation alters surface curvature in Arabidopsis leaves by perturbing the patterns of surface expansion and cell division

The leaf surface usually stays flat, maintained by coordinated growth. Growth perturbation can introduce overall surface curvature, which can be negative, giving a saddle-shaped leaf, or positive, giving a cup-like leaf. Little is known about the molecular mechanisms that underlie leaf flatness, primarily because only a few mutants with altered surface curvature have been isolated and studied. Characterization of mutants of the CINCINNATA-like TCP genes in Antirrhinum and Arabidopsis have revealed that their products help maintain flatness by balancing the pattern of cell proliferation and surface expansion between the margin and the central zone during leaf morphogenesis. On the other hand, deletion of two homologous PEAPOD genes causes cup-shaped leaves in Arabidopsis due to excess division of dispersed meristemoid cells. Here, we report the isolation and characterization of an Arabidopsis mutant, tarani (tni), with enlarged, cup-shaped leaves. Morphometric analyses showed that the positive curvature of the tni leaf is linked to excess growth at the centre compared to the margin. By monitoring the dynamic pattern of CYCLIN D3;2 expression, we show that the shape of the primary arrest front is strongly convex in growing tni leaves, leading to excess mitotic expansion synchronized with excess cell proliferation at the centre. Reduction of cell proliferation and of endogenous gibberellic acid levels rescued the tni phenotype. Genetic interactions demonstrated that TNI maintains leaf flatness independent of TCPs and PEAPODs.

Correct biological timing in Arabidopsis requires multiple light-signaling pathways.

Circadian oscillators provide rhythmic temporal cues for a range of biological processes in plants and animals, enabling anticipation of the day/night cycle and enhancing fitness-associated traits. We have used engineering models to understand the control principles of a plant's response to seasonal variation. We show that the seasonal changes in the timing of circadian outputs require light regulation via feed-forward loops, combining rapid light-signaling pathways with entrained circadian oscillators. Linear time-invariant models of circadian rhythms were computed for 3,503 circadian-regulated genes and for the concentration of cytosolic-free calcium to quantify the magnitude and timing of regulation by circadian oscillators and light-signaling pathways. Bioinformatic and experimental analysis show that rapid light-induced regulation of circadian outputs is associated with seasonal rephasing of the output rhythm. We identify that external coincidence is required for rephasing of multiple output rhythms, and is therefore important in general phase control in addition to specific photoperiod-dependent processes such as flowering and hypocotyl elongation. Our findings uncover a fundamental design principle of circadian regulation, and identify the importance of rapid light-signaling pathways in temporal control.

Absence of branches from xylan in Arabidopsis gux mutants reveals potential for simplification of lignocellulosic biomass.

As one of the most abundant polysaccharides on Earth, xylan will provide more than a third of the sugars for lignocellulosic biofuel production when using grass or hardwood feedstocks. Xylan is characterized by a linear β(1,4)-linked backbone of xylosyl residues substituted by glucuronic acid, 4-O-methylglucuronic acid or arabinose, depending on plant species and cell types. The biological role of these decorations is unclear, but they have a major influence on the properties of the polysaccharide. Despite the recent isolation of several mutants with reduced backbone, the mechanisms of xylan synthesis and substitution are unclear. We identified two Golgi-localized putative glycosyltransferases, GlucUronic acid substitution of Xylan (GUX)-1 and GUX2 that are required for the addition of both glucuronic acid and 4-O-methylglucuronic acid branches to xylan in Arabidopsis stem cell walls. The gux1 gux2 double mutants show loss of xylan glucuronyltransferase activity and lack almost all detectable xylan substitution. Unexpectedly, they show no change in xylan backbone quantity, indicating that backbone synthesis and substitution can be uncoupled. Although the stems are weakened, the xylem vessels are not collapsed, and the plants grow to normal size. The xylan in these plants shows improved extractability from the cell wall, is composed of a single monosaccharide, and requires fewer enzymes for complete hydrolysis. These findings have implications for our understanding of the synthesis and function of xylan in plants. The results also demonstrate the potential for manipulating and simplifying the structure of xylan to improve the properties of lignocellulose for bioenergy and other uses.

The circadian oscillator gene GIGANTEA mediates a long-term response of the Arabidopsis thaliana circadian clock to sucrose.

Circadian clocks are 24-h timing devices that phase cellular responses; coordinate growth, physiology, and metabolism; and anticipate the day-night cycle. Here we report sensitivity of the Arabidopsis thaliana circadian oscillator to sucrose, providing evidence that plant metabolism can regulate circadian function. We found that the Arabidopsis circadian system is particularly sensitive to sucrose in the dark. These data suggest that there is a feedback between the molecular components that comprise the circadian oscillator and plant metabolism, with the circadian clock both regulating and being regulated by metabolism. We used also simulations within a three-loop mathematical model of the Arabidopsis circadian oscillator to identify components of the circadian clock sensitive to sucrose. The mathematical studies identified GIGANTEA (GI) as being associated with sucrose sensing. Experimental validation of this prediction demonstrated that GI is required for the full response of the circadian clock to sucrose. We demonstrate that GI acts as part of the sucrose-signaling network and propose this role permits metabolic input into circadian timing in Arabidopsis.

Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding beta-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic beta-amylase encoding genes in pgi1 leaves, which was accompanied by increased beta-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P) H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.

Early response to nanoparticles in the Arabidopsis transcriptome compromises plant defence and root-hair development through salicylic acid signalling

Background: The impact of nano-scaled materials on photosynthetic organisms needs to be evaluated. Plants represent the largest interface between the environment and biosphere, so understanding how nanoparticles affect them is especially relevant for environmental assessments. Nanotoxicology studies in plants allude to quantum size effects and other properties specific of the nano-stage to explain increased toxicity respect to bulk compounds. However, gene expression profiles after exposure to nanoparticles and other sources of environmental stress have not been compared and the impact on plant defence has not been analysed. Results: Arabidopsis plants were exposed to TiO2-nanoparticles, Ag-nanoparticles, and multi-walled carbon nanotubes as well as different sources of biotic (microbial pathogens) or abiotic (saline, drought, or wounding) stresses. Changes in gene expression profiles and plant phenotypic responses were evaluated. Transcriptome analysis shows similarity of expression patterns for all plants exposed to nanoparticles and a low impact on gene expression compared to other stress inducers. Nanoparticle exposure repressed transcriptional responses to microbial pathogens, resulting in increased bacterial colonization during an experimental infection. Inhibition of root hair development and transcriptional patterns characteristic of phosphate starvation response were also observed. The exogenous addition of salicylic acid prevented some nano-specific transcriptional and phenotypic effects, including the reduction in root hair formation and the colonization of distal leaves by bacteria. Conclusions: This study integrates the effect of nanoparticles on gene expression with plant responses to major sources of environmental stress and paves the way to remediate the impact of these potentially damaging compounds through hormonal priming.