Sessile plants reprogram their metabolic and developmental processes during adaptation to prolonged environmental stresses. To understand the molecular mechanisms underlying adaptation of plant cells to saline stress, we established callus suspension cell cultures from Arabidopsis roots adapted to high salt for an extended period of time. Adapted cells exhibit enhanced salt tolerance compared with control cells. Moreover, acquired salt tolerance is maintained even after the stress is relieved, indicating the existence of a memory of acquired salt tolerance during mitotic cell divisions, known as mitotic stress memory. Metabolite profiling using 1H-nuclear magnetic resonance (NMR) spectroscopy revealed metabolic discrimination between control, salt-adapted and stress-memory cells. Compared with control cells, salt-adapted cells accumulated higher levels of sugars, amino acids and intermediary metabolites in the shikimate pathway, such as coniferin. Moreover, adapted cells acquired thicker cell walls with higher lignin contents, suggesting the importance of adjustments of physical properties during adaptation to elevated saline conditions. When stress-memory cells were reverted to normal growth conditions, the levels of metabolites again readjusted. Whereas most of the metabolic changes reverted to levels intermediate between salt-adapted and control cells, the amounts of sugars, alanine, γ-aminobutyric acid and acetate further increased in stress-memory cells, supporting a view of their roles in mitotic stress memory. Our results provide insights into the metabolic adjustment of plant root cells during adaptation to saline conditions as well as pointing to the function of mitotic memory in acquired salt tolerance.

Korean fir ( Abies koreana ), a rare species endemic to South Korea, is sensitive to climate change. Here, we used next-generation massively parallel sequencing technology and de novo transcriptome assembly to gain a comprehensive overview of the Korean fir transcriptome under heat stress. Sequencing control and heat-treated samples of Korean fir, we obtained more than 194,872,650 clean reads from each sample. After de novo assembly and quantitative assessment, 42,056 unigenes were generated with an average length of 908 bp. In total, 6,401 differentially expressed genes were detected, of which 2,958 were up-regulated and 3,443 down-regulated, between the heat-treated and control samples. A gene ontology analysis of these unigenes revealed heat-stress-related terms, such as “response to stimulus”. Further, in depth analysis revealed 204 transcription factors and 189 Hsps as differentially expressed. Finally, 12 regulated candidate genes associated with heat stress were examined using quantitative real-time PCR (qRT–PCR). In this study, we present the first comprehensive characterisation of Korean fir subjected to heat stress using transcriptome analysis. It provides an important resource for future studies of Korean fir with the objective of identifying heat stress tolerant lines.

i Ptant Breeding and Geneties Research Laberatony, Japan 7bbacco lhc, 700 Higashibara, 7byoda, lwata, Shizuoka. 438-08a2 Japan 2 Department ofBiochemistry, The Uhiversity ofArizona. Biosciences PV'est, Tucson, AZ 85Z2i-O088, U.S.-tl, i Dopartment ofPlant Sciences, 77ie University ofArizona, Biosciences PYOst. Tleicson. AZ 85nl-O088, U.S.A. 4 Department ofMblecular and CeilularBiotogy, 7:he Uitiversicy ofArizona. Biosciences PVes4 Tlicson. .tlZ 85ni-O088, U.S.A.

We have compared rnetabolic effects of high salinity between plants and cell suspension cultures frorn the facultative halophyte Mlesembt:yanthemum castallinum (common ice plant). This plant shows developrnentally-programmed inducibility for a switch from C3-photosynthesis to CAM (Crassulacean Acid Metabolism). The metabolic switch is enhanced by environrnental factors such as drought, low temperature, and, most effectively, soil salinity. CAM induction is dependent on organized leaf tissue and cannot be elicited by salt stress in suspension culture cells. In contrast, the accumulation of proline [Thomas et al. (1992) Plant RhysioL 98: 6as] is induced by NaCl in cultured cells as well as in plants and must be considered a cellular response to stress. We have extended our observations to include another trait of saltand low-temperature-stress responses in the ice plant, the accumulation of putative osmoprotective sugars and sugar alcohols. In whole plants the cyclic sugar alcohol, pinitol, accumulates to amounts that approach 1 M during stress, while in suspension cells no increase in sugar alcohols is observed. The distribution of carbon to different sugars is markedly different be ween cells and plants under stress. Particularly obvious is the distinction between cell types in the different composition of sugars and

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A crucial prerequisite for plant growth and survival is the maintenance of potassium uptake, especially when high sodium surrounds the root zone. The Arabidopsis HIGH-AFFINITY K+ TRANSPORTER1 (HKT1), and its homologs in other salt-sensitive dicots, contributes to salinity tolerance by removing Na+ from the transpiration stream. However, TsHKT1; 2, one of three HKT1 copies in Thellungiella salsuginea, a halophytic Arabidopsis relative, acts as a K+ transporter in the presence of Na+ in yeast (Saccharomyces cerevisiae). Amino-acid sequence comparisons indicated differences between TsHKT1; 2 and most other published HKT1 sequences with respect to an Asp residue (D207) in the second pore-loop domain. Two additional T. salsuginea and most other HKT1 sequences contain Asn (N) in this position. Wild-type TsHKT1; 2 and altered AtHKT1 (AtHKT1(N-D)) complemented K+-uptake deficiency of yeast cells. Mutant hkt1-1 plants complemented with both AtHKT1(N-D) and TsHKT1; 2 showed higher tolerance to salt stress than lines complemented by the wild-type AtHKT1. Electrophysiological analysis in Xenopus laevis oocytes confirmed the functional properties of these transporters and the differential selectivity for Na+ and K+ based on the N/D variance in the pore region. This change also dictated inward-rectification for Na+ transport. Thus, the introduction of Asp, replacing Asn, in HKT1-type transporters established altered cation selectivity and uptake dynamics. We describe one way, based on a single change in a crucial protein that enabled some crucifer species to acquire improved salt tolerance, which over evolutionary time may have resulted in further changes that ultimately facilitated colonization of saline habitats.

Mesembryanthemum crystallinum (ice plant) exhibits extreme tolerance to salt. Epidermal bladder cells (EBCs), developing on the surface of aerial tissues and specialized in sodium sequestration and other protective functions, are critical for the plant's stress adaptation. We present the first transcriptome analysis of EBCs isolated from intact plants, to investigate cell type-specific responses during plant salt adaptation. We developed a de novo assembled, nonredundant EBC reference transcriptome. Using RNAseq, we compared the expression patterns of the EBC-specific transcriptome between control and salt-treated plants. The EBC reference transcriptome consists of 37 341 transcript-contigs, of which 7% showed significantly different expression between salt-treated and control samples. We identified significant changes in ion transport, metabolism related to energy generation and osmolyte accumulation, stress signalling, and organelle functions, as well as a number of lineage-specific genes of unknown function, in response to salt treatment. The salinity-induced EBC transcriptome includes active transcript clusters, refuting the view of EBCs as passive storage compartments in the whole-plant stress response. EBC transcriptomes, differing from those of whole plants or leaf tissue, exemplify the importance of cell type-specific resolution in understanding stress adaptive mechanisms.

Gene co-expression networks provide an important tool for systems biology studies. Using microarray data from the ArrayExpress database, we constructed an Arabidopsis gene co-expression network, termed AtGGM2014, based on the graphical Gaussian model, which contains 102,644 co-expression gene pairs among 18,068 genes. The network was grouped into 622 gene co-expression modules. These modules function in diverse house-keeping, cell cycle, development, hormone response, metabolism, and stress response pathways. We developed a tool to facilitate easy visualization of the expression patterns of these modules either in a tissue context or their regulation under different treatment conditions. The results indicate that at least six modules with tissue-specific expression pattern failed to record modular regulation under various stress conditions. This discrepancy could be best explained by the fact that experiments to study plant stress responses focused mainly on leaves and less on roots, and thus failed to recover specific regulation pattern in other tissues. Overall, the modular structures revealed by our network provide extensive information to generate testable hypotheses about diverse plant signaling pathways. AtGGM2014 offers a constructive tool for plant systems biology studies.

YUCCA (YUC) proteins constitute a family of flavin monooxygenases (FMOs), with an important role in auxin (IAA) biosynthesis. Here we report that Arabidopsis plants overexpressing YUC6 display enhanced IAA-related phenotypes and exhibit improved drought stress tolerance, low rate of water loss and controlled ROS accumulation under drought and oxidative stresses. Co-overexpression of an IAA-conjugating enzyme reduces IAA levels but drought stress tolerance is unaffected, indicating that the stress-related phenotype is not based on IAA overproduction. YUC6 contains a previously unrecognized FAD- and NADPH-dependent thiol-reductase activity (TR) that overlaps with the FMO domain involved in IAA biosynthesis. Mutation of a conserved cysteine residue (Cys-85) preserves FMO but suppresses TR activity and stress tolerance, whereas mutating the FAD- and NADPH-binding sites, that are common to TR and FMO domains, abolishes all outputs. We provide a paradigm for a single protein playing a dual role, regulating plant development and conveying stress defence responses.

The Arabidopsis-related model species (ARMS) Thellungiella salsuginea and Thellungiella parvula have generated broad interest in salt stress research. While general growth characteristics of these species are similar to Arabidopsis, some aspects of their life cycle require particular attention in order to obtain healthy plants, with a large production of seeds in a relatively short time. This chapter describes basic procedures for growth, maintenance, and Agrobacterium-mediated transformation of ARMS. Where appropriate, differences in requirements between Thellungiella spp. and Arabidopsis are highlighted, along with basic growth requirements of other less studied candidate model species. Current techniques for comparative genomics analysis between Arabidopsis and ARMS are also described in detail.

Comprehending the reasons for the natural variation and distribution of life-forms in different environments has been a major motivational force for biology since antiquity. With Mendel's discoveries, Darwin's natural selection theories became armed with the concept of genotype.How a genotype is inherited, selected, and how this results in a phenotype have become a spirited debate that has ultimately led to our ideas of genetics and epigenetics. The concept of genetic memory that arose from developmental biology has underpinned the desire to understand the molecular basis of epigenetics. This has finally led to uncovering of many of the molecular building blocks and regulatory pathways of epigenetics. The possibility that the environment through epigenetic regulation may direct trait inheritance has even begun to question some of our fundamental paradigms of the function of the environment in shaping evolution. Some direct role of the environment in heritable phenotype variation cannot be ruled out now. This has blurred our concepts of transgenerational and developmental genetics so much that understanding the connections between these processes has taken an enormously important status in biology. The pace of growth of our knowledge of the biochemical participants in the epigenetic informational system has been truly impressive. Major epigenetic marks involve covalent chemical modifications to DNA and to histones. How these modifications and noncoding RNAs are organized into an epigenetic informational system is being intensively investigated.Understanding the way in which the environment controls the connections and use of epigenetic marks with the DNA sequence code promises to expose a rich new world of biology and new toolboxes that will afford stunning new capabilities in agriculture, medicine, and all practical applications of biology. This chapter discusses the sea change in our understanding of genome environment interactions.

Schrenkiella parvula (formerly Thellungiella parvula), a close relative of Arabidopsis (Arabidopsis thaliana) and Brassica crop species, thrives on the shores of Lake Tuz, Turkey, where soils accumulate high concentrations of multiple-ion salts. Despite the stark differences in adaptations to extreme salt stresses, the genomes of S. parvula and Arabidopsis show extensive synteny. S. parvula completes its life cycle in the presence of Na+, K+, Mg2(+), Li+, and borate at soil concentrations lethal to Arabidopsis. Genome structural variations, including tandem duplications and translocations of genes, interrupt the colinearity observed throughout the S. parvula and Arabidopsis genomes. Structural variations distinguish homologous gene pairs characterized by divergent promoter sequences and basal-level expression strengths. Comparative RNA sequencing reveals the enrichment of ion-transport functions among genes with higher expression in S. parvula, while pathogen defense-related genes show higher expression in Arabidopsis. Key stress-related ion transporter genes in S. parvula showed increased copy number, higher transcript dosage, and evidence for subfunctionalization. This extremophyte offers a framework to identify the requisite adjustments of genomic architecture and expression control for a set of genes found in most plants in a way to support distinct niche adaptation and lifestyles.

Genome sequences and RNA-seq data provide unparalleled views into the genetic basis that underlies plant phenotypes. This chapter outlines a paradigm shift in plant biology originating from the relative ease of generating sequences, and points to tools that have enabled global transcript analyses (RNA-seq), replacing arduous microarray hybridization experiments and laborious quantitative RT-PCR platform designs. Soon, it can be envisioned that one may replace individual transcript assays by measuring how the entire transcriptome responds globally to a manipulation or mutation. The chapter advances a position that the knowledge of genome content and structure, and conditional transcript expression characteristics can explain a plant's growth and adaptation to the environment. This point is highlighted by comparing the genome and transcriptome of the abiotic stress-sensitive Arabidopsis thaliana with its close relatives, Thellungiella parvula and Thellungiella salsuginea.

Our knowledge base involving the biochemical participants of epigenetic control has expanded greatly over the last decade. The role of epigenetic marks to DNA and histones controlled by non-coding RNAs is one of the most intensely studied areas of biology today. This review covers many of themechanisms that non-coding RNAs and other molecules use to control gene expression and eventually affect responses to the environment. In the first part of the review, we discuss the array of covalent modifications to the genome that constitute the epigenome, which consists of the histone variants, covalent modifications, and post-translational modifications that result in gene expression changes. How the histone variants and post-translationalmodifications including, acetylation, methylation, phosphorylation, ubiquitination and sumoylation help form the epigenome is also summarized. Our eventual understanding of how the environment controls these modifications will open incredible opportunities in agriculture, medicine and the development of practical tools for biology. In the second part of this review we discuss the growing list of environmentally-mediated epigenetic-modifications, and examples of transgenerational epigenetic inheritance events, that may begin to change our views of adaptive responses to the environment and evolution.

In the interaction between plants and pathogens, carbon (C) resources provide energy and C skeletons to maintain, among many functions, the plant immune system. However, variations in C availability on pathogen associated molecular pattern (PAMP) triggered immunity (PTI) have not been systematically examined. Here, three types of starch mutants with enhanced susceptibility to Pseudomonas syringae pv. tomato DC3000 hrcC were examined for PTI. In a dark period-dependent manner, the mutants showed compromised induction of a PTI marker, and callose accumulation in response to the bacterial PAMP flagellin, flg22. In combination with weakened PTI responses in wild type by inhibition of the TCA cycle, the experiments determined the necessity of C-derived energy in establishing PTI. Global gene expression analyses identified flg22 responsive genes displaying C supply-dependent patterns. Nutrient recycling-related genes were regulated similarly by C-limitation and flg22, indicating re-arrangements of expression programs to redirect resources that establish or strengthen PTI. Ethylene and NAC transcription factors appear to play roles in these processes. Under C-limitation, PTI appears compromised based on suppression of genes required for continued biosynthetic capacity and defenses through flg22. Our results provide a foundation for the intuitive perception of the interplay between plant nutrition status and pathogen defense.

Understanding of gene regulatory networks requires discovery of expression modules within gene co-expression networks and identification of promoter motifs and corresponding transcription factors that regulate their expression. A commonly used method for this purpose is a top-down approach based on clustering the network into a range of densely connected segments, treating these segments as expression modules, and extracting promoter motifs from these modules. Here, we describe a novel bottom-up approach to identify gene expression modules driven by known cis-regulatory motifs in the gene promoters. For a specific motif, genes in the co-expression network are ranked according to their probability of belonging to an expression module regulated by that motif. The ranking is conducted via motif enrichment or motif position bias analysis. Our results indicate that motif position bias analysis is an effective tool for genome-wide motif analysis. Sub-networks containing the top ranked genes are extracted and analyzed for inherent gene expression modules. This approach identified novel expression modules for the G-box, W-box, site II, and MYB motifs from an Arabidopsis thaliana gene co-expression network based on the graphical Gaussian model. The novel expression modules include those involved in house-keeping functions, primary and secondary metabolism, and abiotic and biotic stress responses. In addition to confirmation of previously described modules, we identified modules that include new signaling pathways. To associate transcription factors that regulate genes in these co-expression modules, we developed a novel reporter system. Using this approach, we evaluated MYB transcription factor-promoter interactions within MYB motif modules.

Although a role for microRNA399 (miR399) in plant responses to phosphate (Pi) starvation has been indicated, the regulatory mechanism underlying miR399 gene expression is not clear. Here, we report that AtMYB2 functions as a direct transcriptional activator for miR399 in Arabidopsis (Arabidopsis thaliana) Pi starvation signaling. Compared with untransformed control plants, transgenic plants constitutively overexpressing AtMYB2 showed increased miR399f expression and tissue Pi contents under high Pi growth and exhibited elevated expression of a subset of Pi starvation-induced genes. Pi starvation-induced root architectural changes were more exaggerated in AtMYB2-overexpressing transgenic plants compared with the wild type. AtMYB2 directly binds to a MYB-binding site in the miR399f promoter in vitro, as well as in vivo, and stimulates miR399f promoter activity in Arabidopsis protoplasts. Transcription of AtMYB2 itself is induced in response to Pi deficiency, and the tissue expression patterns of miR399f and AtMYB2 are similar. Both genes are expressed mainly in vascular tissues of cotyledons and in roots. Our results suggest that AtMYB2 regulates plant responses to Pi starvation by regulating the expression of the miR399 gene.

Environmental challenges to plants typically entail retardation of vegetative growth and delay or cessation of flowering. Here we report a link between the flowering time regulator, GIGANTEA (GI), and adaptation to salt stress that is mechanistically based on GI degradation under saline conditions, thus retarding flowering. GI, a switch in photoperiodicity and circadian clock control, and the SNF1-related protein kinase SOS2 functionally interact. In the absence of stress, the GI: SOS2 complex prevents SOS2-based activation of SOS1, the major plant Na+/H+-antiporter mediating adaptation to salinity. GI overexpressing, rapidly flowering, plants show enhanced salt sensitivity, whereas gi mutants exhibit enhanced salt tolerance and delayed flowering. Salt-induced degradation of GI confers salt tolerance by the release of the SOS2 kinase. The GI-SOS2 interaction introduces a higher order regulatory circuit that can explain in molecular terms, the long observed connection between floral transition and adaptive environmental stress tolerance in Arabidopsis.