Iron (Fe) homeostasis is integrated with the production of Reactive Oxygen Species (ROS) whose distribution at the root tip participates in the control of root growth. Excess Fe increases ferritin abundance, enabling the storage of Fe which contributes to protection of plants against Fe-induced oxidative stress. AtFer1 and AtFer3 are the two ferritin genes expressed in the meristematic zone, pericycle and endodermis of the Arabidopsis thaliana (Arabidopsis) root, and it is in these regions that we observe Fe stained dots. This staining disappears in the triple fer1-3-4 ferritin mutant. Fe excess decreases primary root length in the same way in wild-type and in fer1-3-4 mutant. In contrast, the Fe mediated decrease of lateral root (LR) length and density is enhanced in fer1-3-4 plants due to a defect in LR emergence. We observe that this interaction between excess Fe, ferritin and RSA is in part mediated by the H2O2/O2 (.-) balance between the root cell proliferation and differentiation zones regulated by the UPB1 transcription factor. Further, meristem size is also decreased in response to Fe excess in ferritin mutant plants, implicating cell cycle arrest mediated by the ROS-activated SMR5/SMR7 cyclin-dependent kinase inhibitors pathway in the interaction between Fe and RSA.
Plastid-to-nucleus signaling is essential for the coordination and adjustment of cellular metabolism in response to environmental and developmental cues of plant cells. A variety of operational retrograde signaling pathways have been described that are thought to be triggered by reactive oxygen species, photosynthesis redox imbalance, tetrapyrrole intermediates, and other metabolic traits. Here we report a meta-analysis based on transcriptome and protein interaction data. Comparing the output of these pathways reveals the commonalities and peculiarities stimulated by six different sources impinging on operational retrograde signaling. Our study provides novel insights into the interplay of these pathways, supporting the existence of an as-yet unknown core response module of genes being regulated under all conditions tested. Our analysis further highlights affiliated regulatory cis-elements and classifies abscisic acid and auxin-based signaling as secondary components involved in the response cascades following a plastidial signal. Our study provides a global analysis of structure and interfaces of different pathways involved in plastid-to-nucleus signaling and a new view on this complex cellular communication network.
Natural illumination conditions are highly variable and because of their sessile life style plantsare forced to acclimate to them at cellular and molecular level. Changes in light intensity orquality induce changes in the reduction/oxidation (redox) state of the photosynthetic electronchain that act as trigger for compensatory acclimation responses comprising functional andstructural adjustments of photosynthesis and metabolism. Such responses include redoxcontrolledchanges in plant gene expression in nucleus and organelles. Here we describe astrategy for the identification of early redox-regulated genes (ERGs) in the nucleus of themodel organism Arabidopsis thaliana which significantly respond 30 or 60 min after thegeneration of a reduction signal in the photosynthetic electron transport chain. By comparingthe response of wild-type plants with that of the acclimation mutant stn7 we could specificallyidentify ERGs. The results reveal a significant impact of chloroplast redox signals on distinctnuclear gene groups including genes for the mitochondrial electron transport chain,tetrapyrrole biosynthesis, carbohydrate metabolism and signalling lipid synthesis. Theseexpression profiles are clearly different from that observed in response to reduction of thephotosynthetic electron transport (PET) by high light treatments. The identified ERGs, thus,are unique to redox imbalances in PET and were used for the analysis of potential redoxresponsivecis-elements, trans-factors and chromosomal regulatory hot spots. The dataidentify a novel redox-responsive element and indicate extensive redox control attranscriptional and chromosomal levels that point to an unprecedented impact of redox signalson epigenetic processes.
Dear Editor, Seed germination is controlled by multiple endo genous and environmental factors, which are integrated to trigger this developmental process at the right time. Gibberellins (GAs) are known to induce this process, and the levels of GAs are modulated by light—one of the most important environmental factors affecting seed germina tion. The bHLH transcription factor PIL5 (PHYTOCHROME INTERACTING FACTOR 3LIKE 5) is the master repressor of lightmediated seed germination in Arabidopsis (Oh et al., 2004). In seeds kept in the dark, PIL5 activates transcrip tion of the GAI (GA INSENSITIVE) gene (Peng et al., 1997; Oh et al., 2007), a DELLA transcriptional regulator that represses GAmediated processes (Sun, 2011). GAI plays a role in many growth processes with both unique and overlapping functions with another DELLA protein: RGA (REPRESSOR OF ga1-3) (Dill and Sun, 2001). Also, the DOF transcription factor DAG1 (DOF AFFECTING GERMINATION1) acts in the lightmediated seed germination pathway downstream of PIL5: DAG1 expression is reduced in seeds irradiated for 24 h with red (R) light, and this reduction is dependent on PIL5 as, in pil5 mutant seeds, DAG1 expression is reduced irrespective of light conditions (Gabriele et al., 2010). Null mutant seeds dag1 need a fluence rate six times lower than wildtype to germinate (Papi et al., 2000, 2002); similarly, gai–t6rga28 double mutant seeds require less R light fluences than wildtype ones to germinate (Oh et al., 2007). To further clarify the role of DAG1 in lightmediated seed germination, we focus here on the functional rela tionship between DAG1 and GAI in the control of this process. We have recently demonstrated that DAG1 specifi cally represses AtGA3ox1 expression. In dag1 mutant seeds, only this GA biosynthetic gene was upregulated, while the level of expression of AtGA3ox2 and AtGA2ox2 were unchanged compared to the wildtype (Gabriele et al., 2010). A very similar expression profile of AtGA3ox1 was shown by Oh et al. (2007) in gai–t6rga28 double mutant seeds. To verify whether GAI plays a role in the regulation of GA metabolic genes, and in particular of AtGA3ox1, we performed a quantitative RT–PCR (RT–qPCR) analysis on gai-t6 mutant seeds. The level of the AtGA3ox1 transcript was highly increased in the gai-t6 null mutant compared to the wildtype, both in seeds imbibed in the dark and those exposed to R light (Figure 1A), while expression of AtGA3ox2 and AtGA2ox2 was not significantly altered. Since—similarly to DAG1 inactivation—GAI inactivation specifically affected AtGA3ox1 expression, we decided to verify whether the presence of GAI is necessary for DAG1 mediated repression of AtGA3ox1. In agreement with our hypothesis, promoter analysis of GAIregulated genes revealed a significant enrichment of DOFbinding sites (GallegoBartolomé et al., 2011), suggesting that these transcription factors may mediate GAI activity. We used the dag1DAG1–HA (Gabriele et al., 2010) and the dag1gai-t6DAG1–HA lines, which overexpress DAG1 respectively in the dag1 and dag1gai-t6 mutant backgrounds. Both these lines expressed the DAG1–HA chimeric protein as revealed by immunoblot analysis (Supplemental Figure 1). As expected, the expression of AtGA3ox1 in dag1DAG1–HA seeds was highly reduced compared to wildtype both in the dark and under R light, due to overexpression of DAG1–HA, whereas AtGA3ox1 was strongly overexpressed in dag1gai-t6DAG1–HA seeds (Figure 1B and 1C), suggesting that both DAG1 and GAI are involved in the regulation of AtGA3ox1. Since inactivation of GAI makes DAG1 unable to repress AtGA3ox1 expression, we set to assess whether these two factors directly collaborate in regulating this GA biosynthetic gene. We performed chromatin immunopre cipitation (ChIP) assays using the GAI–MYC transgenic line constructed by Oh et al. (2007), and the dag1DAG1–HA line (Gabriele et al., 2010) as a positive control. Crosslinked and sonicated protein–DNA complexes were precipitated with antiMYC and antiHA antibodies, respectively. We amplified by realtime PCR (qPCR) three regions of the AtGA3ox1 promoter containing different numbers of cop ies of DOFbinding sites (0, 2, and 15) (Figure 1D). As a nega tive control, we performed the same assays without adding the antibody, or with both antibodies on wildtype seeds (Supplemental Figure 2). The relative amounts of precipi tated promoter fragments of AtGA3ox1 by DAG1–HA are higher than the negative control, and the enrichment of the target fragment is proportional to the number of DOF sites present in the region. By contrast, the enrichment of precipitated promoter fragments of AtGA3ox1 was very
Isoprenoids are functionally and structurally the most diverse group of plant metabolites reported to date. They can function as primary metabolites, participating in essential plant cellular processes, and as secondary metabolites, of which many have substantial commercial, pharmacological, and agricultural value. Isoprenoid end products participate in plants in a wide range of physiological processes acting in them both synergistically, such as chlorophyll and carotenoids during photosynthesis, or antagonistically, such as gibberellic acid and abscisic acid during seed germination. It is therefore expected that fluxes via isoprenoid metabolic network are tightly controlled both temporally and spatially, and that this control occurs at different levels of regulation and in an orchestrated manner over the entire isoprenoid metabolic network. In this review, we summarize our current knowledge of the topology of the plant isoprenoid pathway network and its regulation at the gene expression level following diver
Secondary cell walls provide plants with rigidity and strength to support their body weight and ensure water and nutrient transport. They also provide textiles, timber, and potentially second-generation biofuels for human use. Genes responsible for synthesis of the different cell wall components, namely cellulose, hemicelluloses, and lignin, are coordinately expressed and under transcriptional regulation. In the past several years, cell wall-related NAC and MYB transcription factors have been intensively investigated in different species and shown to be master switches of secondary cell wall biosynthesis. Positive and negative regulators, which function upstream of NAC master switches, have also been identified in different plant tissues. Further elucidation of the regulatory mechanisms of cell wall synthesis will facilitate the engineering of plant feedstocks suitable for biofuel production.
To better understand the response of rice to nutrient stress, we have taken a systematic approach to identify rice genes that respond to deficiency of macronutrients and affect rice growth. We report here the expression and bio- logical functions of a previously uncharacterized rice gene that we have named NRR （nutrition response and root growth）. NRR is alternatively spliced, producing two 5＇-coterminal transcripts, NRRa and NRRb, encoding two proteins of 308 and 223 aa, respectively. Compared to NRRb, NRRa possesses an additional CCT domain at the C-terminus. Expression of NRR in rice seedling roots was significantly influenced by deficiency of macronutrients. Knock-down of expression of NRRa or NRRb by RNA interference resulted in enhanced rice root growth. By contrast, overexpression of NRRa in rice exhibited significantly retarded root growth. These results revealed that both NRRa and NRRb played negative regulatory roles in rice root growth. Our findings suggest that NRRa and NRRb, acting as the key com
Radially arranged cortical microtubules are a prominent feature of guard cells. Guard cells expressing GFP- tubulin showed consistent changes in the appearance of microtubules when stomata opened or closed. Guard cells showed fewer microtubule structures as stomata closed, whether induced by transfer to darkness, ABA, hydrogen per- oxide, or sodium hydrogen carbonate. Guard cells kept in the dark （closed stomata） showed increases in microtubule struc- tures and stomatal aperture on light treatment. GFP-EB1, marking microtubule growing plus ends, showed no change in number of plus ends or velocity of assembly on stomatal closure. Since the number of growing plus ends and the rate of plus-end growth did not change when microtubule structure numbers declined, microtubule instability and/or rearrange- ment must be responsible for the apparent loss of microtubules. Guard cells with closed stomata showed more cytosolic GFP-fluorescence than those with open stomata as cortical microtubules became disassembled, altho
Spectro-microscopy, a combination of fluorescence microscopy with spatially resolved spectroscopic techni- ques, provides new and exciting tools for functional cell biology in living organisms. This review focuses on recent devel- opments in spectro-microscopic applications for the investigation of living plant cells in their native tissue context. The application of spectro-microscopic methods led to the recent discovery of a fast signal response pathway for the brassi- nosteroide receptor BRI1 in the plasma membrane of living plant cells. Moreover, the competence of different plant cell types to respond to environmental or endogenous stimuli was determined in vivo by correlation analysis of different optical and spectroscopic readouts such as fluorescence lifetime （FLT）. Furthermore, a new spectro-microscopic technique, fluorescence intensity decay shape analysis microscopy （FIDSAM）, has been developed. FIDSAM is capable of imaging low- expressed fluorophore-tagged proteins at high spatial resolution and pr
The circadian clock temporally coordinates plant growth and metabolism in close synchronization with the diurnal and seasonal environmental changes. Research over the last decade has identified a number of clock components and a variety of regulatory mechanisms responsible for the rhythmic oscillations in metabolic and physiological activities. At the core of the clock, transcriptional/translational feedback loops modulate the expression of a significant proportion of the genome. In this article, we briefly describe some of the very recent advances that have improved our understanding of clock organization and function in Arabidopsis thaliana. The new studies illustrate the role of clock protein complex for- mation on circadian gating of plant growth and identify alternative splicing as a new regulatory mechanism for clock function. Examination of key clock properties such as temperature compensation has also opened new avenues for func- tional research within the plant clockwork. The emerging connections bet
Anthocyanins are synthesized in the cytosolic surface of the endoplasmic reticulum （ER） but dominantly accumulate in the vacuole. Little is known about how anthocyanins are transported from the ER to the vacuole. Here, we provide evidence supporting that Transparent Testa 19 （TT19）, a glutathione 5-transferase （GST）, functions as a carrier to transport cyanidin and/or anthocyanins to the tonoplast. We identified a novel tt19 mutant （tt19-7）, which barely accumulates anthocyanins but produces a 36% higher level of flavonol than the wild-type （WT）, from ethyl methanesulfonate mutagenized seeds. Expressing TT19-fused green fluorescence protein （GFP） in tt19-7 rescues the mutant phenotype in defective anthocyanin biosynthesis, indicating that TT19-GFP is functional. We further showed that TT19-GFP is localized not only in the cytoplasm and nuclei, but also on the tonoplast. The membrane localization of TT19-GFP was further ascertained by immunoblot analysis. In vitro assay showed that the purified recombinant TT1
Phytochromes in seed plants are known to move into nuclei in a red light-dependent manner with or without interacting factors. Here, we show phytochrome relocation to the nuclear region in phytochrome-dependent Adiantum capillus-veneris spore germination by partial spore-irradiation experiments. The nuclear or non-nuclear region of imbibed spores was irradiated with a microbeam of red and/or far-red light and the localization of phytochrome involved in spore germination was estimated from the germination rate. The phytochrome for spore germination existed throughout whole spore under darkness after imbibition, but gradually migrated to the nuclear region following red light irradiation. In- tracellular distribution of PHY-GUS fusion proteins expressed in germinated spores by particle bombardment showed the migration of Acphy2, but not Acphyl, into nucleus in a red light-dependent manner, suggesting that Acphy2 is the photoreceptor for fern spore germination.
Organization of proteins into complexes is crucial for many cellular functions. Recently, the SUT1 protein was shown to form homodimeric complexes, to be associated with lipid raft-like microdomains in yeast as well as in plants and to undergo endocytosis in response to brefeldin A. We therefore aimed to identify SUTl-interacting proteins that might be involved in dimerization, endocytosis, or targeting of SUT1 to raft-like microdomains. Therefore, we identified potato membrane proteins, which are associated with the detergent-resistant membrane （DRM） fraction. Among the proteins identified, we clearly confirmed StSUT1 as part of DRM in potato source leaves. We used the yeast two-hybrid split ubiq- uitin system （SUS） to systematically screen for interaction between the sucrose transporter StSUT1 and other membrane- associated or soluble proteins in vivo. The SUS screen was followed by immunoprecipitation using affinity-purified StSUTl-specific peptide antibodies and mass spectrometric analysis of co-precipita
Integration of the genetic and metabolic fingerprinting can provide a new approach to differentiate similar Traditional Chinese Medical （TCM） materials. Two leguminous plants, Mojia Huangqi and Menggu Huangqi, are important medical herbs and share great similarities in morphology, chemical constituent, and genomic DNA sequence. The taxonomy of Mojia Huangqi and Menggu Huangqi has been debated for more than 50 years and discrimination of TCM materials directly affects the pharmacological and clinical effects. AFLP based genetic fingerprinting and GC-TOF/MS-based meta- bolic fingerprinting were used to successfully discriminate the two species. The results of AFLP supported the opinion that Menggu Huangqi was a variant of Mojia Huangqi. The metabolic fingerprinting showed growth locations have greater impacts on the metabolite composition and quantity than the genotypes （cultivated versus wild） in Menggu Huangqi. The difference of some soluble sugars, fatty acids, proline, and polyamine reflected plant adaptati
Plants constantly survey the surrounding environment using several sets of photoreceptors. They can sense changes in the quantity （= intensity） and quality （=wavelength） of light and use this information to adjust their physiological responses, growth, and developmental patterns. In addition to the classical photoreceptors, such as phytochromes, crypto- chromes, and phototropins, ZEITLUPE （ZTL）, FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 （FKF1）, and LOV KELCH PROTEIN 2 （LKP2） proteins have been recently identified as blue-light photoreceptors that are important for regulation of the circadian clock and photoperiodic flowering. The ZTL/FKF1/LKP2 protein family possesses a unique combination of domains： a blue-light-absorb- ing LOV （Light, Oxygen, or Voltage） domain along with domains involved in protein degradation. Here, we summarize recent advances in our understanding of the function of the Arabidopsis ZTL/FKF1/LKP2 proteins. We summarize the distinct pho- tochemical properties of their LOV domains and discuss t
Despite extensive study, the molecular structure of the chromophore-binding pocket of phytochrome A （phyA）, the principal photoreceptor controlling photomorphogenesis in plants, has not yet been successfully resolved. Here, we report a series of two-dimensional （2-D） magic-angle spinning solid-state NMR experiments on the recombi- nant N-terminal, 65-kDa PAS-GAF-PHY light-sensing module of phytochrome A3 from oat （Avena sativa）, assembled with uniformly 13C- and lSN-labeled phycocyanobilin （u-[13C,15N]-PCB-As.phyA3）. The Pr state of this protein was studied regarding the electronic structure of the chromophore and its interactions with the proximal amino acids. Using 2-D 13C-13C and 1HJSN experiments, a complete set of 13C and 15N assignments for the chromophore were obtained. Also, a large number of 1H-13C distance restraints between the chromophore and its binding pocket were revealed by inter- facial heteronuclear correlation spectroscopy. 13C doublings of the chromophore A-ring region and the C-ring carbo
Photomorphogenesis is controlled by multiple signaling pathways, including the light and brassinosteroid （BR） pathways. BR signaling activates the BZR1 transcription factor, which is required for suppressing photomorphogen- esis in the dark, We identified a suppressor of the BR hypersensitive mutant bzrl-lD and named it bzrl-lD suppressorl- Dominant （bzsl-D）. The bzsl-D mutation was caused by overexpression of a B-box zinc finger protein BZS1, which is transcriptionally repressed by BZR1. Overexpression of BZS1 causes de-etiolation in the dark, short hypocotyls in the light, reduced sensitivity to BR treatment, and repression of many BR-activated genes. Knockdown of BZS1 by co-suppression partly suppressed the short hypocotyl phenotypes of BR-deficient or insensitive mutants. These results support that BZSl is a negative regulator of BR response. BZS1 overexpressors are hypersensitive to different wavelengths of light and loss of function of BZS1 reduces plant sensitivity to light and partly suppresses the co
Light is emerging as a central regulator of plant immune responses against herbivores and pathogens. Solar UVoB radiation plays an important role as a positive modulator of plant defense. However, since UV-B photons can interact with a wide spectrum of molecular targets in plant tissues, the mechanisms that mediate their effects on plant defense have remained elusive. Here, we show that ecologically meaningful doses of UV-B radiation increase Arabidopsis resis- tance to the necrotrophic fungus Botrytis cinerea and that this effect is mediated by the photoreceptor UVR8. The UV-B effect on plant resistance was conserved in mutants impaired in jasmonate （JA） signaling （jar1-1 and P35S：JAZlO.4） or metabolism of tryptophan-derived defense compounds （pen2-1, pacl3-1, pen2 pad3）, suggesting that neither regulation of the JA pathway nor changes in levels of indolic glucosinolates （iGS） or camalexin are involved in this response. UV-B radiation, acting through UVR8, increased the levels of flavonoids and sinapates in