Transcriptomes of Eight Arabidopsis thaliana Accessions Reveal Core Conserved, Genotype- and Organ-Specific Responses to Flooding Stress.

Climate change has increased the frequency and severity of flooding events, with significant negative impact on agricultural productivity. These events often submerge plant aerial organs and roots, limiting growth and survival due to a severe reduction in light reactions and gas exchange necessary for photosynthesis and respiration, respectively. To distinguish molecular responses to the compound stress imposed by submergence, we investigated transcriptomic adjustments to darkness in air and under submerged conditions using eight Arabidopsis (Arabidopsis thaliana) accessions differing significantly in sensitivity to submergence. Evaluation of root and rosette transcriptomes revealed an early transcriptional and posttranscriptional response signature that was conserved primarily across genotypes, although flooding susceptibility-associated and genotype-specific responses also were uncovered. Posttranscriptional regulation encompassed darkness- and submergence-induced alternative splicing of transcripts from pathways involved in the alternative mobilization of energy reserves. The organ-specific transcriptome adjustments reflected the distinct physiological status of roots and shoots. Root-specific transcriptome changes included marked up-regulation of chloroplast-encoded photosynthesis and redox-related genes, whereas those of the rosette were related to the regulation of development and growth processes. We identified a novel set of tolerance genes, recognized mainly by quantitative differences. These included a transcriptome signature of more pronounced gluconeogenesis in tolerant accessions, a response that included stress-induced alternative splicing. This study provides organ-specific molecular resolution of genetic variation in submergence responses involving interactions between darkness and low-oxygen constraints of flooding stress and demonstrates that early transcriptome plasticity, including alternative splicing, is associated with the ability to cope with a compound environmental stress.

Group VII ethylene response factor diversification and regulation in four species from flood-prone environments.

Flooding events negatively affect plant performance and survival. Flooding gradients thereby determine the dynamics in vegetation composition and species abundance. In adaptation to flooding, the group VII Ethylene Response Factor genes (ERF-VIIs) play pivotal roles in rice and Arabidopsis through regulation of anaerobic gene expression and antithetical survival strategies. We investigated if ERF-VIIs have a similar role in mediating survival strategies in eudicot species from flood-prone environments. Here, we studied the evolutionary origin and regulation of ERF-VII transcript abundance and the physiological responses in species from two genera of divergent taxonomic lineages (Rumex and Rorippa). Synteny analysis revealed that angiosperm ERF-VIIs arose from two ancestral loci and that subsequent diversification and duplication led to the present ERF-VII variation. We propose that subtle variation in the regulation of ERF-VII transcript abundance could explain variation in tolerance among Rorippa species. In Rumex, the main difference in flood tolerance correlated with the genetic variation in ERF-VII genes. Large transcriptional differences were found by comparing the two genera: darkness and dark submergence-induced Rumex ERF-VIIs, whereas HRE2 expression was increased in submerged Rorippa roots. We conclude that the involvement of ERF-VIIs in flooding tolerance developed in a phylogenetic-dependent manner, with subtle variations within taxonomic clades.

cDNA library preparation.

The construction of full-length cDNA libraries allows researchers to study gene expression and protein interactions and undertake gene discovery. Recent improvements allow the construction of high-quality cDNA libraries, with small amounts of mRNA. In parallel, these improvements allow for the incorporation of adapters into the cDNA, both at the 5' and 3' end of the cDNA. The 3' adapter is attached to the oligo-dT primer that is used by the reverse transcriptase, whereas the 5' adapter is incorporated by the template switching properties of the MMLV reverse transcriptase. This allows directional cloning and eliminates inefficient steps like adapter ligation, phosphorylation, and methylation. Another important step in the construction of high-quality cDNA libraries is the normalization. The difference in the levels of expression between genes might be several orders of magnitude. Therefore, it is essential that the cDNA library is normalized. With a recently discovered enzyme, duplex-specific nuclease, it is possible to normalize the cDNA library, based on the fact that more abundant molecules are more likely to reanneal after denaturation compared to rare molecules.

TaMYB13-1, a R2R3 MYB transcription factor, regulates the fructan synthetic pathway and contributes to enhanced fructan accumulation in bread wheat.

Fructans are the major component of temporary carbon reserve in the stem of temperate cereals, which is used for grain filling. Three families of fructosyltransferases are directly involved in fructan synthesis in the vacuole of Triticum aestivum. The regulatory network of the fructan synthetic pathway is largely unknown. Recently, a sucrose-upregulated wheat MYB transcription factor (TaMYB13-1) was shown to be capable of activating the promoter activities of sucrose:sucrose 1-fructosyltransferase (1-SST) and sucrose:fructan 6-fructosyltransferase (6-SFT) in transient transactivation assays. This work investigated TaMYB13-1 target genes and their influence on fructan synthesis in transgenic wheat. TaMYB13-1 overexpression resulted in upregulation of all three families of fructosyltransferases including fructan:fructan 1-fructosyltransferase (1-FFT). A γ-vacuolar processing enzyme (γ-VPE1), potentially involved in processing the maturation of fructosyltransferases in the vacuole, was also upregulated by TaMYB13-1 overexpression. Multiple TaMYB13 DNA-binding motifs were identified in the Ta1-FFT1 and Taγ-VPE1 promoters and were bound strongly by TaMYB13-1. The expression profiles of these target genes and TaMYB13-1 were highly correlated in recombinant inbred lines and during stem development as well as the transgenic and non-transgenic wheat dataset, further supporting a direct regulation of these genes by TaMYB13-1. TaMYB13-1 overexpression in wheat led to enhanced fructan accumulation in the leaves and stems and also increased spike weight and grain weight per spike in transgenic plants under water-limited conditions. These data suggest that TaMYB13-1 plays an important role in coordinated upregulation of genes necessary for fructan synthesis and can be used as a molecular tool to improve the high fructan trait.

TAF13 interacts with PRC2 members and is essential for Arabidopsis seed development.

TBP-Associated Factors (TAFs) are components of complexes like TFIID, TFTC, SAGA/STAGA and SMAT that are important for the activation of transcription, either by establishing the basic transcription machinery or by facilitating histone acetylation. However, in Drosophila embryos several TAFs were shown to be associated with the Polycomb Repressive Complex 1 (PRC1), even though the role of this interaction remains unclear. Here we show that in Arabidopsis TAF13 interacts with MEDEA and SWINGER, both members of a plant variant of Polycomb Repressive Complex 2 (PRC2). PRC2 variants play important roles during the plant life cycle, including seed development. The taf13 mutation causes seed defects, showing embryo arrest at the 8-16 cell stage and over-proliferation of the endosperm in the chalazal region, which is typical for Arabidopsis PRC2 mutants. Our data suggest that TAF13 functions together with PRC2 in transcriptional regulation during seed development.

Dissecting the molecular basis of the contribution of source strength to high fructan accumulation in wheat.

Fructans represent the major component of water soluble carbohydrates (WSCs) in the maturing stem of temperate cereals and are an important temporary carbon reserve for grain filling. To investigate the importance of source carbon availability in fructan accumulation and its molecular basis, we performed comparative analyses of WSC components and the expression profiles of genes involved in major carbohydrate metabolism and photosynthesis in the flag leaves of recombinant inbred lines from wheat cultivars Seri M82 and Babax (SB lines). High sucrose levels in the mature flag leaf (source organ) were found to be positively associated with WSC and fructan concentrations in both the leaf and stem of SB lines in several field trials. Analysis of Affymetrix expression array data revealed that high leaf sucrose lines grown in abiotic-stress-prone environments had high expression levels of a number of genes in the leaf involved in the sucrose synthetic pathway and photosynthesis, such as Calvin cycle genes, antioxidant genes involved in chloroplast H(2)O(2) removal and genes involved in energy dissipation. The expression of the majority of genes involved in fructan and starch synthetic pathways were positively correlated with sucrose levels in the leaves of SB lines. The high level of leaf fructans in high leaf sucrose lines is likely attributed to the elevated expression levels of fructan synthetic enzymes, as the mRNA levels of three fructosyltransferase families were consistently correlated with leaf sucrose levels among SB lines. These data suggest that high source strength is one of the important genetic factors determining high levels of WSC in wheat.

TaMYB13 is a transcriptional activator of fructosyltransferase genes involved in ß-2,6-linked fructan synthesis in wheat.

Fructans are soluble fructosyl-oligosaccharides deposited in many cool-season grass species as a carbon reserve; they are synthesised by fructosyltransferases. In wheat and barley fructans can accumulate in mature stems at a very high level and serve as an important carbon source for grain filling. Fructan synthesis in temperate cereals is regulated by sucrose level and developmental signals, and functions as a metabolic adjustment for carbon balance between carbon supply and sink demand. In this study the expression levels of a highly homologous group of Triticum aestivumMYB genes (TaMYB13-1, TaMYB13-2 and TaMYB13-3) were found to be positively correlated with the mRNA levels of sucrose:sucrose 1-fructosyltransferase (1-SST) and sucrose:fructan 6-fructosyltransferase (6-SFT) in wheat stems among recombinant inbred lines with a wide range of fructan concentrations through Affymetrix array expression analysis. This expression correction extended to expression profiles during stem development. TaMYB13 contains an R2R3-type MYB domain. In vitro random DNA-binding site selection followed by base substitution mutagenesis revealed that TaMYB13 bound to a (A/G/T)TT(A/T/C)GGT core sequence, which was present in the promoters of wheat Ta1-SST and Ta6-SFT genes as well as a barley Hv6-SFT gene. Transactivation analysis showed that TaMYB13 was a transcriptional activator and could markedly enhance the expression of 1-SST and 6-SFT promoter-driven reporter genes in wheat. Elimination of TaMYB13-binding sites in Ta6-SFT and Ta1-SST promoters markedly reduced TaMYB13-mediated reporter gene transactivation. These data suggest that TaMYB13 and its orthologues are positive regulators for controlling the expression of major fructosyltransferases involved in the fructan synthetic pathway in temperate cereals.

Overlapping and antagonistic activities of BASIC PENTACYSTEINE genes affect a range of developmental processes in Arabidopsis.

The BASIC PENTACYSTEINE (BPC) proteins are a plant-specific transcription factor family that is present throughout land plants. The Arabidopsis BPC proteins have been categorized into three classes based on sequence similarity, and we demonstrate that there is functional overlap between classes. Single gene mutations produce no visible phenotypic effects, and severe morphological phenotypes occur only in higher order mutants between members of classes I and II, with the most severe phenotype observed in bpc1-1 bpc2 bpc4 bpc6 plants. These quadruple mutants are dwarfed and display small curled leaves, aberrant ovules, altered epidermal cells and reduced numbers of lateral roots. Affected processes include coordinated growth of cell layers, cell shape determination and timing of senescence. Disruption of BPC3 function rescues some aspects of the bpc1-1 bpc2 bpc4 bpc6 phenotype, indicating that BPC3 function may be antagonistic to other members of the family. Ethylene response is diminished in bpc1-1 bpc2 bpc4 bpc6 plants, although not all aspects of the phenotype can be explained by reduced ethylene sensitivity. Our data indicate that the BPC transcription factor family is integral for a wide range of processes that support normal growth and development.

Reversible male sterility in eggplant (Solanum melongena L.) by artificial microRNA-mediated silencing of general transcription factor genes.

Since decades, plant male sterility is considered a powerful tool for biological containment to minimize unwanted self-pollination for hybrid seed production. Furthermore, prevention of pollen dispersal also answers to concerns regarding transgene flow via pollen from Genetically Modified (GM) crops to traditional crop fields or wild relatives. We induced male sterility by suppressing endogenous general transcription factor genes, TAFs, using anther-specific promoters combined with artificial microRNA (amiRNA) technology (Schwab et al., 2006). The system was made reversible by the ethanol inducible expression of an amiRNA-insensitive form of the target gene. We provide proof of concept in eggplant, a cultivated crop belonging to the Solanaceae family that includes many important food crops. The transgenic eggplants that we generated are completely male sterile and fertility can be fully restored by short treatments with ethanol, confirming the efficiency but also the reliability of the system in view of open field cultivation. By combining this system with induced parthenocarpy (Rotino et al., 1997), we provide a novel example of complete transgene containment in eggplant, which enables biological mitigation measures for the benefit of coexistence or biosafety purposes for GM crop cultivation.

The Arabidopsis BET bromodomain factor GTE4 is involved in maintenance of the mitotic cell cycle during plant development.

Bromodomain and Extra Terminal domain (BET) proteins are characterized by the presence of two types of domains, the bromodomain and the extra terminal domain. They bind to acetylated lysines present on histone tails and control gene transcription. They are also well known to play an important role in cell cycle regulation. In Arabidopsis (Arabidopsis thaliana), there are 12 BET genes; however, only two of them, IMBIBITION INDUCIBLE1 and GENERAL TRANSCRIPTION FACTOR GROUP E6 (GTE6), were functionally analyzed. We characterized GTE4 and show that gte4 mutant plants have some characteristic features of cell cycle mutants. Their size is reduced, and they have jagged leaves and a reduced number of cells in most organs. Moreover, cell size is considerably increased in the root, and, interestingly, the root quiescent center identity seems to be partially lost. Cell cycle analyses revealed that there is a delay in activation of the cell cycle during germination and a premature arrest of cell proliferation, with a switch from mitosis to endocycling, leading to a statistically significant increase in ploidy levels in the differentiated organs of gte4 plants. Our results point to a role of GTE4 in cell cycle regulation and specifically in the maintenance of the mitotic cell cycle.

Comprehensive interaction map of the Arabidopsis MADS Box transcription factors.

Interactions between proteins are essential for their functioning and the biological processes they control. The elucidation of interaction maps based on yeast studies is a first step toward the understanding of molecular networks and provides a framework of proteins that possess the capacity and specificity to interact. Here, we present a comprehensive plant protein-protein interactome map of nearly all members of the Arabidopsis thaliana MADS box transcription factor family. A matrix-based yeast two-hybrid screen of >100 members of this family revealed a collection of specific heterodimers and a few homodimers. Clustering of proteins with similar interaction patterns pinpoints proteins involved in the same developmental program and provides valuable information about the participation of uncharacterized proteins in these programs. Furthermore, a model is proposed that integrates the floral induction and floral organ formation networks based on the interactions between the proteins involved. Heterodimers between flower induction and floral organ identity proteins were observed, which point to (auto)regulatory mechanisms that prevent the activity of flower induction proteins in the flower.

BASIC PENTACYSTEINE1, a GA binding protein that induces conformational changes in the regulatory region of the homeotic Arabidopsis gene SEEDSTICK.

The mechanisms for the regulation of homeotic genes are poorly understood in most organisms, including plants. We identified BASIC PENTACYSTEINE1 (BPC1) as a regulator of the homeotic Arabidopsis thaliana gene SEEDSTICK (STK), which controls ovule identity, and characterized its mechanism of action. A combination of tethered particle motion analysis and electromobility shift assays revealed that BPC1 is able to induce conformational changes by cooperative binding to purine-rich elements present in the STK regulatory sequence. Analysis of STK expression in the bpc1 mutant showed that STK is upregulated. Our results give insight into the regulation of gene expression in plants and provide the basis for further studies to understand the mechanisms that control ovule identity in Arabidopsis.

MADS-box protein complexes control carpel and ovule development in Arabidopsis.

The AGAMOUS (AG) gene is necessary for stamen and carpel development and is part of a monophyletic clade of MADS-box genes that also includes SHATTERPROOF1 (SHP1), SHP2, and SEEDSTICK (STK). Here, we show that ectopic expression of either the STK or SHP gene is sufficient to induce the transformation of sepals into carpeloid organs bearing ovules. Moreover, the fact that these organ transformations occur when the STK gene is expressed ectopically in ag mutants shows that STK can promote carpel development in the absence of AG activity. We also show that STK, AG, SHP1, and SHP2 can form multimeric complexes and that these interactions require the SEPALLATA (SEP) MADS-box proteins. We provide genetic evidence for this role of the SEP proteins by showing that a reduction in SEP activity leads to the loss of normal ovule development, similar to what occurs in stk shp1 shp2 triple mutants. Together, these results indicate that the SEP proteins, which are known to form multimeric complexes in the control of flower organ identity, also form complexes to control normal ovule development.