HD-Zip II transcription factors control distal stem cell fate in Arabidopsis roots by linking auxin signaling to the FEZ/SOMBRERO pathway.

In multicellular organisms, specialized tissues are generated by specific populations of stem cells through cycles of asymmetric cell divisions, where one daughter undergoes differentiation and the other maintains proliferative properties. In Arabidopsis thaliana roots, the columella - a gravity-sensing tissue that protects and defines the position of the stem cell niche - represents a typical example of a tissue whose organization is exclusively determined by the balance between proliferation and differentiation. The columella derives from a single layer of stem cells through a binary cell fate switch that is precisely controlled by multiple, independent regulatory inputs. Here, we show that the HD-Zip II transcription factors (TFs) HAT3, ATHB4 and AHTB2 redundantly regulate columella stem cell fate and patterning in the Arabidopsis root. The HD-Zip II TFs promote columella stem cell proliferation by acting as effectors of the FEZ/SMB circuit and, at the same time, by interfering with auxin signaling to counteract hormone-induced differentiation. Overall, our work shows that HD-Zip II TFs connect two opposing parallel inputs to fine-tune the balance between proliferation and differentiation in columella stem cells.

DRT111/SFPS Splicing Factor Controls Abscisic Acid Sensitivity during Seed Development and Germination.

RNA splicing is a fundamental mechanism contributing to the definition of the cellular protein population in any given environmental condition. DNA-DAMAGE REPAIR/TOLERATION PROTEIN111 (DRT111)/SPLICING FACTOR FOR PHYTOCHROME SIGNALING is a splicing factor previously shown to interact with phytochrome B and characterized for its role in splicing of pre-mRNAs involved in photomorphogenesis. Here, we show that DRT111 interacts with Arabidopsis (Arabidopsis thaliana) Splicing Factor1, involved in 3' splicing site recognition. Double- and triple-mutant analysis shows that DRT111 controls splicing of ABI3 and acts upstream of the splicing factor SUPPRESSOR OF ABI3-ABI5. DRT111 is highly expressed in seeds and stomata of Arabidopsis and is induced by long-term treatments of polyethylene glycol and abscisic acid (ABA). DRT111 knock-out mutants are defective in ABA-induced stomatal closure and are hypersensitive to ABA during seed germination. Conversely, DRT111 overexpressing plants show ABA-hyposensitive seed germination. RNA-sequencing experiments show that in dry seeds, DRT111 controls expression and splicing of genes involved in osmotic-stress and ABA responses, light signaling, and mRNA splicing, including targets of ABSCISIC ACID INSENSITIVE3 (ABI3) and PHYTOCHROME INTERACTING FACTORs (PIFs). Consistently, expression of the germination inhibitor SOMNUS, induced by ABI3 and PIF1, is upregulated in imbibed seeds of drt111-2 mutants. Together, these results indicate that DRT111 controls sensitivity to ABA during seed development, germination, and stomatal movements, and integrates ABA- and light-regulated pathways to control seed germination.

The PP2A-interactor TIP41 modulates ABA responses in Arabidopsis thaliana.

Modulation of growth in response to environmental cues is a fundamental aspect of plant adaptation to abiotic stresses. TIP41 (TAP42 INTERACTING PROTEIN OF 41 kDa) is the Arabidopsis thaliana orthologue of proteins isolated in mammals and yeast that participate in the Target-of-Rapamycin (TOR) pathway, which modifies cell growth in response to nutrient status and environmental conditions. Here, we characterized the function of TIP41 in Arabidopsis. Expression analyses showed that TIP41 is constitutively expressed in vascular tissues, and is induced following long-term exposure to NaCl, polyethylene glycol and abscisic acid (ABA), suggesting a role of TIP41 in adaptation to abiotic stress. Visualization of a fusion protein with yellow fluorescent protein indicated that TIP41 is localized in the cytoplasm and the nucleus. Abolished expression of TIP41 results in smaller plants with a lower number of rosette leaves and lateral roots, and an increased sensitivity to treatments with chemical TOR inhibitors, indicating that TOR signalling is affected in these mutants. In addition, tip41 mutants are hypersensitive to ABA at germination and seedling stage, whereas over-expressing plants show higher tolerance. Several TOR- and ABA-responsive genes are differentially expressed in tip41, including iron homeostasis, senescence and ethylene-associated genes. In yeast and mammals, TIP41 provides a link between the TOR pathway and the protein phosphatase 2A (PP2A), which in plants participates in several ABA-mediated mechanisms. Here, we showed an interaction of TIP41 with the catalytic subunit of PP2A. Taken together, these results offer important insights into the function of Arabidopsis TIP41 in the modulation of plant growth and ABA responses.

Arabidopsis HD-Zip II proteins regulate the exit from proliferation during leaf development in canopy shade.

The shade avoidance response is mainly evident as increased plant elongation at the expense of leaf and root expansion. Despite the advances in understanding the mechanisms underlying shade-induced hypocotyl elongation, little is known about the responses to simulated shade in organs other than the hypocotyl. In Arabidopsis, there is evidence that shade rapidly and transiently reduces the frequency of cell division in young first and second leaf primordia through a non-cell-autonomous mechanism. However, the effects of canopy shade on leaf development are likely to be complex and need to be further investigated. Using combined methods of genetics, cell biology, and molecular biology, we uncovered an effect of prolonged canopy shade on leaf development. We show that persistent shade determines early exit from proliferation in the first and second leaves of Arabidopsis. Furthermore, we demonstrate that the early exit from proliferation in the first and second leaves under simulated shade depends at least in part on the action of the Homeodomain-leucine zipper II (HD-Zip II) transcription factors ARABIDOPSIS THALIANA HOMEOBOX2 (ATHB2) and ATHB4. Finally, we provide evidence that the ATHB2 and ATHB4 proteins work in concert. Together the data contribute new insights on the mechanisms controlling leaf development under canopy shade.

Multiple Links between HD-Zip Proteins and Hormone Networks.

HD-Zip proteins are unique to plants, and contain a homeodomain closely linked to a leucine zipper motif, which are involved in dimerization and DNA binding. Based on homology in the HD-Zip domain, gene structure and the presence of additional motifs, HD-Zips are divided into four families, HD-Zip I⁻IV. Phylogenetic analysis of HD-Zip genes using transcriptomic and genomic datasets from a wide range of plant species indicate that the HD-Zip protein class was already present in green algae. Later, HD-Zips experienced multiple duplication events that promoted neo- and sub-functionalizations. HD-Zip proteins are known to control key developmental and environmental responses, and a growing body of evidence indicates a strict link between members of the HD-Zip II and III families and the auxin machineries. Interactions of HD-Zip proteins with other hormones such as brassinolide and cytokinin have also been described. More recent data indicate that members of different HD-Zip families are directly involved in the regulation of abscisic acid (ABA) homeostasis and signaling. Considering the fundamental role of specific HD-Zip proteins in the control of key developmental pathways and in the cross-talk between auxin and cytokinin, a relevant role of these factors in adjusting plant growth and development to changing environment is emerging.

Arabidopsis HD-Zip II transcription factors control apical embryo development and meristem function.

The Arabidopsis genome encodes ten Homeodomain-Leucine zipper (HD-Zip) II proteins. ARABIDOPSIS THALIANA HOMEOBOX 2 (ATHB2), HOMEOBOX ARABIDOPSIS THALIANA 1 (HAT1), HAT2, HAT3 and ATHB4 are regulated by changes in the red/far red light ratio that induce shade avoidance in most of the angiosperms. Here, we show that progressive loss of HAT3, ATHB4 and ATHB2 activity causes developmental defects from embryogenesis onwards in white light. Cotyledon development and number are altered in hat3 athb4 embryos, and these defects correlate with changes in auxin distribution and response. athb2 gain-of-function mutation and ATHB2 expression driven by its promoter in hat3 athb4 result in significant attenuation of phenotypes, thus demonstrating that ATHB2 is functionally redundant to HAT3 and ATHB4. In analogy to loss-of-function mutations in HD-Zip III genes, loss of HAT3 and ATHB4 results in organ polarity defects, whereas triple hat3 athb4 athb2 mutants develop one or two radialized cotyledons and lack an active shoot apical meristem (SAM). Consistent with overlapping expression pattern of HD-Zip II and HD-Zip III gene family members, bilateral symmetry and SAM defects are enhanced when hat3 athb4 is combined with mutations in PHABULOSA (PHB), PHAVOLUTA (PHV) or REVOLUTA (REV). Finally, we show that ATHB2 is part of a complex regulatory circuit directly involving both HD-Zip II and HD-Zip III proteins. Taken together, our study provides evidence that a genetic system consisting of HD-Zip II and HD-Zip III genes cooperates in establishing bilateral symmetry and patterning along the adaxial-abaxial axis in the embryo as well as in controlling SAM activity.

Untargeted Metabolomics Reveals Predominant Alterations in Lipid Metabolism Following Light Exposure in Broccoli Sprouts.

The consumption of vegetables belonging to the family Brassicaceae (e.g., broccoli and cauliflower) is linked to a reduced incidence of cancer and cardiovascular diseases. The molecular composition of such plants is strongly affected by growing conditions. Here we developed an unbiased metabolomics approach to investigate the effect of light and dark exposure on the metabolome of broccoli sprouts and we applied such an approach to provide a bird's-eye view of the overall metabolic response after light exposure. Broccoli seeds were germinated and grown hydroponically for five days in total darkness or with a light/dark photoperiod (16 h light/8 h dark cycle). We used an ultra-performance liquid-chromatography system coupled to an ion-mobility, time-of-flight mass spectrometer to profile the large array of metabolites present in the sprouts. Differences at the metabolite level between groups were analyzed using multivariate statistical analyses, including principal component analysis and correlation analysis. Altered metabolites were identified by searching publicly available and in-house databases. Metabolite pathway analyses were used to support the identification of subtle but significant changes among groups of related metabolites that may have gone unnoticed with conventional approaches. Besides the chlorophyll pathway, light exposure activated the biosynthesis and metabolism of sterol lipids, prenol lipids, and polyunsaturated lipids, which are essential for the photosynthetic machinery. Our results also revealed that light exposure increased the levels of polyketides, including flavonoids, and oxylipins, which play essential roles in the plant's developmental processes and defense mechanism against herbivores. This study highlights the significant contribution of light exposure to the ultimate metabolic phenotype, which might affect the cellular physiology and nutritional value of broccoli sprouts. Furthermore, this study highlights the potential of an unbiased omics approach for the comprehensive study of the metabolism.

Dynamics of the shade-avoidance response in Arabidopsis.

Shade-intolerant plants perceive the reduction in the ratio of red light (R) to far-red light (FR) as a warning of competition with neighboring vegetation and display a suite of developmental responses known as shade avoidance. In recent years, major progress has been made in understanding the molecular mechanisms underlying shade avoidance. Despite this, little is known about the dynamics of this response and the cascade of molecular events leading to plant adaptation to a low-R/FR environment. By combining genome-wide expression profiling and computational analyses, we show highly significant overlap between shade avoidance and deetiolation transcript profiles in Arabidopsis (Arabidopsis thaliana). The direction of the response was dissimilar at the early stages of shade avoidance and congruent at the late ones. This latter regulation requires LONG HYPOCOTYL IN FAR RED1/SLENDER IN CANOPY SHADE1 and phytochrome A, which function largely independently to negatively control shade avoidance. Gene network analysis highlights a subnetwork containing ELONGATED HYPOCOTYL5 (HY5), a master regulator of deetiolation, in the wild type and not in phytochrome A mutant upon prolonged low R/FR. Network analysis also highlights a direct connection between HY5 and HY5 HOMOLOG (HYH), a gene functionally implicated in the inhibition of hypocotyl elongation and known to be a direct target of the HY5 transcription factor. Kinetics analysis show that the HYH gene is indeed late induced by low R/FR and that its up-regulation depends on the action of HY5, since it does not occur in hy5 mutant. Therefore, we propose that one way plants adapt to a low-R/FR environment is by enhancing HY5 function.

Exploring the metabolic and physiological roles of HQT in S. lycopersicum by gene editing.

The most abundant phenolic compound in Solanaceous plants is chlorogenic acid (CGA), which possesses protective properties such as antimicrobial and antioxidant activities. These properties are particularly relevant when plants are under adverse conditions, such as pathogen attack, excess light, or extreme temperatures that cause oxidative stress. Additionally, CGA has been shown to absorb UV-B light. In tomato and potato, CGA is mainly produced through the HQT pathway mediated by the enzyme hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase. However, the absence of natural or induced mutants of this gene has made it unclear whether other pathways contribute to CGA production and accumulation. To address this question, we used CRISPR technology to generate multiple knock-out mutant lines in the tomato HQT gene. The resulting slhqt plants did not accumulate CGA or other caffeoylquinic acids (CQAs) in various parts of the plant, indicating that CQA biosynthesis depends almost entirely on the HQT pathway in tomato and, likely, other Solanaceous crops. We also found that the lack of CGA in slhqt plants led to higher levels of hydroxycinnamoyl-glucose and flavonoids compared to wild-type plants. Gene expression analysis revealed that this metabolic reorganization was partly due to flux redirection, but also involved modulation of important transcription factor genes that regulate secondary metabolism and sense environmental conditions. Finally, we investigated the physiological role of CGA in tomato and found that it accumulates in the upper epidermis where it acts as a protector against UV-B irradiation.

Coordination of biradial-to-radial symmetry and tissue polarity by HD-ZIP II proteins.

Symmetry establishment is a critical process in the development of multicellular organs and requires careful coordination of polarity axes while cells actively divide within tissues. Formation of the apical style in the Arabidopsis gynoecium involves a bilateral-to-radial symmetry transition, a stepwise process underpinned by the dynamic distribution of the plant morphogen auxin. Here we show that SPATULA (SPT) and the HECATE (HEC) bHLH proteins mediate the final step in the style radialisation process and synergistically control the expression of adaxial-identity genes, HOMEOBOX ARABIDOPSIS THALIANA 3 (HAT3) and ARABIDOPSIS THALIANA HOMEOBOX 4 (ATHB4). HAT3/ATHB4 module drives radialisation of the apical style by promoting basal-to-apical auxin flow and via a negative feedback mechanism that finetune auxin distribution through repression of SPT expression and cytokinin sensitivity. Thus, this work reveals the molecular basis of axes-coordination and hormonal cross-talk during the sequential steps of symmetry transition in the Arabidopsis style.

ATHB2 is a negative regulator of germination in Arabidopsis thaliana seeds.

The germination timing of seeds is of the utmost adaptive importance for plant populations. Light is one of the best characterized factors promoting seed germination in several species. The germination is also finely regulated by changes in hormones levels, mainly those of gibberellin (GA) and abscisic acid (ABA). Here, we performed physiological, pharmacological, and molecular analyses to uncover the role of ATHB2, an HD-ZIP II transcription factor, in germination of Arabidopsis seeds. Our study demonstrated that ATHB2 is a negative regulator and sustains the expression of transcription factors to block germination promoted by light. Besides, we found that ATHB2 increases ABA sensitivity. Moreover, ABA and auxin content in athb2-2 mutant is higher than wild-type in dry seeds, but the differences disappeared during the imbibition in darkness and the first hours of exposition to light, respectively. Some ABA and light transcription factors are up-regulated by ATHB2, such as ABI5, ABI3, XERICO, SOMNUS and PIL5/PIF1. In opposition, PIN7, an auxin transport, is down-regulated. The role of ATHB2 as a repressor of germination induced by light affecting the gemination timing, could have differential effects on the establishment of seedlings altering the competitiveness between crops and weeds in the field.

Identification of a novel cis-regulatory element for UV-B-induced transcription in Arabidopsis.

Ultraviolet-B light (UV-B) regulates the expression of genes in a wavelength- and fluence rate-dependent fashion. A signaling pathway consisting of CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1) and UV RESISTANCE LOCUS 8 (UVR 8) mediates responsiveness to longer wavelength, low intensity UV-B light-activating, for example, HY5 gene expression. By contrast, transcription of another group of genes, including ANAC13, modulated by shorter wavelength, higher intensity UV-B is controlled by a yet unknown and largely COP1-independent signaling cascade. Here we provide evidence by promoter deletion analysis, and characterization of genetic mutants displaying aberrant expression patterns, that two cis-regulatory elements, designated MRE(ANAC13) and UVBox(ANAC13), are required for maximal UV-B induction of the ANAC13 gene in transgenic plants. These elements are located in the proximal 150-bp region of the ANAC13 promoter. They show no significant similarity to each other; the putative MRE(ANAC13) (-AACCTT-) is closely related to MRE(CHS) (-AACCTA-) found in the CHALCONE SYNTHASE (CHS) gene, whereas UVBox(ANAC13) (with core sequence CAAG) represents a novel cis-regulatory element. The novel UVBox(ANAC13) sequence is significantly enriched in the promoter region of a subset of UV-B-induced genes with similar activation properties as ANAC13. In addition, we demonstrate that expression of a chimeric gene containing only the dimerized 12-mer containing UVBox(ANAC13) fused to a minimal CaMV35S promoter/luciferase reporter is (i) efficiently induced by shorter wavelength, higher intensity UV-B, but (ii) does not respond either to longer wavelength UV-B and red light or (iii) to abscisic acid treatment and osmotic, salt, heat and cold stresses.

Embedding mRNA stability in correlation analysis of time-series gene expression data.

Current methods for the identification of putatively co-regulated genes directly from gene expression time profiles are based on the similarity of the time profile. Such association metrics, despite their central role in gene network inference and machine learning, have largely ignored the impact of dynamics or variation in mRNA stability. Here we introduce a simple, but powerful, new similarity metric called lead-lag R(2) that successfully accounts for the properties of gene dynamics, including varying mRNA degradation and delays. Using yeast cell-cycle time-series gene expression data, we demonstrate that the predictive power of lead-lag R(2) for the identification of co-regulated genes is significantly higher than that of standard similarity measures, thus allowing the selection of a large number of entirely new putatively co-regulated genes. Furthermore, the lead-lag metric can also be used to uncover the relationship between gene expression time-series and the dynamics of formation of multiple protein complexes. Remarkably, we found a high lead-lag R(2) value among genes coding for a transient complex.

Salinity and ABA Seed Responses in Pepper: Expression and Interaction of ABA Core Signaling Components.

Abscisic acid (ABA) plays an important role in various aspects of plant growth and development, including adaptation to stresses, fruit development and ripening. In seeds, ABA participates through its core signaling components in dormancy instauration, longevity determination, and inhibition of germination in unfavorable environmental conditions such as high soil salinity. Here, we show that seed germination in pepper was delayed but only marginally reduced by ABA or NaCl with respect to control treatments. Through a similarity search, pepper orthologs of ABA core signaling components PYL (PYRABACTIN RESISTANCE1-LIKE), PP2C (PROTEIN PHOSPHATASE2C), and SnRK2 (SUCROSE NONFERMENTING1 (SNF1)-RELATED PROTEIN KINASE2) genes were identified. Gene expression analyses of selected members showed a low abundance of PYL and SnRK2 transcripts in dry seeds compared to other tissues, and an up-regulation at high concentrations of ABA and/or NaCl for both positive and negative regulators of ABA signaling. As expected, in hydroponically-grown seedlings exposed to NaCl, only PP2C encoding genes were up-regulated. Yeast two hybrid assays performed among putative pepper core components and with Arabidopsis thaliana orthologs confirmed the ability of the identified proteins to function in ABA signaling cascade, with the exception of a CaABI isoform cloned from seeds. BiFC assay in planta confirmed some of the interactions obtained in yeast. Altogether, our results indicate that a low expression of perception and signaling components in pepper seeds might contribute to explain the observed high percentages of seed germination in the presence of ABA. These results might have direct implications on the improvement of seed longevity and vigor, a bottleneck in pepper breeding.

Expression dynamics of the pea rbcS multigene family and organ distribution of the transcripts.

We have determined the nucleotide sequence of two members (rbcS-3A and -3C) of the pea nuclear gene family encoding the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase. Both rbcS-3A and -3C are interrupted by two introns located at the same positions as those of the other three pea rbcS genes. Compared with the other pea rbcS genes the rbcS-3C gene has the most divergent 5'- and 3'-flanking sequences while the rbcS-3A gene has a larger and highly divergent intron 1. All five pea rbcS genes are conserved in their coding regions but show considerable sequence differences in their 3'-untranslated portion. The 3' sequence divergence of the rbcS genes has allowed us to use S1 nuclease mapping procedures to compare their expression levels in different organs and during light induction. All the rbcS genes are differentially expressed in various organs of the pea plants; moreover, specific rbcS transcripts are under-represented in seeds and petals. In leaves there is a 10-fold difference between the highest and lowest specific rbcS transcript levels. By quantitating the distribution of rbcS transcripts during light, phytochrome and blue light induction of immature (etiolated), and mature (green), pea leaves, we show that the genes are differentially activated during leaf development.

Collective behavior in gene regulation: post-transcriptional regulation and the temporal compartmentalization of cellular cycles.

Self-sustained oscillations are perhaps the most studied objects in science. The accomplishment of such a task reliably and accurately requires the presence of specific control mechanisms to face the presence of variable and largely unpredictable environmental stimuli and noise. Self-sustained oscillations of transcript abundance are, in fact, widespread and are not limited to the reproductive cycle but are also observed during circadian rhythms, metabolic cycles, developmental cycles and so on. To date, much of the literature has focused on the transcriptional machinery underlying control of the basic timing of transcript abundance. However, mRNA abundance is known to be regulated at the post-transcriptional level also and the relative contribution of the two mechanisms to gene-expression programmes is currently a major challenge in molecular biology. Here, we review recent results showing the relevance of the post-transcriptional regulation layer and present a statistical reanalysis of the yeast metabolic cycle using publicly available gene-expression and RNA-binding data. Taken together, the recent theoretical and experimental developments reviewed and the results of our reanalysis strongly indicate that regulation of mRNA stability is a widespread, phase-specific and finely tuned mechanism for the multi-layer control of gene expression needed to achieve high flexibility and adaptability to external and internal signals.

Multiple Pathways in the Control of the Shade Avoidance Response.

To detect the presence of neighboring vegetation, shade-avoiding plants have evolved the ability to perceive and integrate multiple signals. Among them, changes in light quality and quantity are central to elicit and regulate the shade avoidance response. Here, we describe recent progresses in the comprehension of the signaling mechanisms underlying the shade avoidance response, focusing on Arabidopsis, because most of our knowledge derives from studies conducted on this model plant. Shade avoidance is an adaptive response that results in phenotypes with a high relative fitness in individual plants growing within dense vegetation. However, it affects the growth, development, and yield of crops, and the design of new strategies aimed at attenuating shade avoidance at defined developmental stages and/or in specific organs in high-density crop plantings is a major challenge for the future. For this reason, in this review, we also report on recent advances in the molecular description of the shade avoidance response in crops, such as maize and tomato, and discuss their similarities and differences with Arabidopsis.

Reduced brain UCP2 expression mediated by microRNA-503 contributes to increased stroke susceptibility in the high-salt fed stroke-prone spontaneously hypertensive rat.

UCP2 maps nearby the lod score peak of STR1-stroke QTL in the SHRSP rat strain. We explored the potential contribution of UCP2 to the high-salt diet (JD)-dependent increased stroke susceptibility of SHRSP. Male SHRSP, SHRSR, two reciprocal SHRSR/SHRSP-STR1/QTL stroke congenic lines received JD for 4 weeks to detect brain UCP2 gene/protein modulation as compared with regular diet (RD). Brains were also analyzed for NF-κB protein expression, oxidative stress level and UCP2-targeted microRNAs expression level. Next, based on knowledge that fenofibrate and Brassica Oleracea (BO) stimulate UCP2 expression through PPARα activation, we monitored stroke occurrence in SHRSP receiving JD plus fenofibrate versus vehicle, JD plus BO juice versus BO juice plus PPARα inhibitor. Brain UCP2 expression was markedly reduced by JD in SHRSP and in the (SHRsr.SHRsp-(D1Rat134-Mt1pa)) congenic line, whereas NF-κB expression and oxidative stress level increased. The opposite phenomenon was observed in the SHRSR and in the (SHRsp.SHRsr-(D1Rat134-Mt1pa)) reciprocal congenic line. Interestingly, the UCP2-targeted rno-microRNA-503 was significantly upregulated in SHRSP and decreased in SHRSR upon JD, with consistent changes in the two reciprocal congenic lines. Both fenofibrate and BO significantly decreased brain microRNA-503 level, upregulated UCP2 expression and protected SHRSP from stroke occurrence. In vitro overexpression of microRNA-503 in endothelial cells suppressed UCP2 expression and led to a significant increase of cell mortality with decreased cell viability. Brain UCP2 downregulation is a determinant of increased stroke predisposition in high-salt-fed SHRSP. In this context, UCP2 can be modulated by both pharmacological and nutraceutical agents. The microRNA-503 significantly contributes to mediate brain UCP2 downregulation in JD-fed SHRSP.

Functional analysis of transcription factors by microparticle bombardment.

This chapter describes a method for rapid gene expression assays in Arabidopsis. Three distinct plasmids are codelivered into leaves by microparticle bombarment. The first one carries a transacting factor gene driven by the cauliflower mosaic virus (CaMV) 35S promoter. The second plasmid includes a reporter luciferase (LUC) gene transcribed from a promoter containing the TF binding site(s). The third plasmid contains a reference beta-glucuronidase (GUS) gene transcribed from the 35S-CaMV promoter, which is used to normalize data derived from independent experiments. This method could be applied to study systematically the functional properties of Arabidopsis transcription factors as well as to identify promoters and control region(s) regulated by specific transcription factors.

Light and shade in the photocontrol of Arabidopsis growth.

Plants have evolved sophisticated sensing mechanisms that operate through phytochromes, perceiving changes in the red:far-red ratio, which trigger morphological changes to avoid shade. The shade-avoidance response essentially redirects resources and growth potential from the leaf and storage organs into increased extension growth to optimize light capture by plants. Recent studies implicate ATHB-2, a homeodomain-leucine zipper transcription factor, as a regulator of shade-avoidance responses and establish a strong link between this factor and auxin signaling. The action of ATHB-2 is likely to generate changes in auxin distribution that produce distinct but coordinated effects on different cell types across the plant. Future studies should highlight how polarity of auxin transport is altered in response to light-quality changes.