The WRKY transcription factors have been reported to function as positive or negative regulators in many different biological processes including plant development, defense regulation and stress ...response. This study isolated a maize WRKY gene, ZmWRKY17, and characterized its role in tolerance to salt stress by generating transgenic Arabidopsis plants. Expression of the ZmWRKY17 was upregulated by drought, salt and abscisic acid (ABA) treatments. ZmWRKY17 was localized in the nucleus with no transcriptional activation in yeast. Yeast one-hybrid assay showed that ZmWRKY17 can specifically bind to W-box, and it can activate W-box-dependent transcription in planta. Heterologous overexpression of ZmWRKY17 in Arabidopsis remarkably reduced plant tolerance to salt stress, as determined through physiological analyses of the cotyledons greening rate, root growth, relative electrical leakage and malondialdehyde content. Additionally, ZmWRKY17 transgenic plants showed decreased sensitivity to ABA during seed germination and early seedling growth. Transgenic plants accumulated higher content of ABA than wild-type (WT) plants under NaCl condition. Transcriptome and quantitative real-time PCR analyses revealed that some stress-related genes in transgenic seedlings showed lower expression level than that in the WT when treated with NaCl. Taken together, these results suggest that ZmWRKY17 may act as a negative regulator involved in the salt stress responses through ABA signalling.
Monogalactosyldiacylglycerol (MGDG) is the major lipid constituent of thylakoid membranes and is essential for chloroplast biogenesis in plants. In Arabidopsis (Arabidopsis thaliana), MGDG is ...predominantly synthesized by inner envelope-localized MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE1 (MGD1); its knockout causes albino seedlings. Because of the lethal phenotype of the null MGD1 mutant, functional details of MGDG synthesis at seedling development have remained elusive. In this study, we used an inducible gene-suppression system to investigate the impact of MGDG synthesis on cotyledon development. We created transgenic Arabidopsis lines that express an artificial microRNA targeting MGD1 (amiR-MGD1) under the control of a dexamethasone-inducible promoter. The induction of amiR-MGDl resulted in up to 75% suppression of MGD1 expression, although the resulting phenotypes related to chloroplast development were diverse, even within a line. The strong MGD1 suppression by continuous dexamethasone treatment caused substantial decreases in galactolipid content in cotyledons, leading to severe defects in the formation of thylakoid membranes and impaired photosynthetic electron transport. Time-course analyses of the MGD1 suppression during seedling germination revealed that MGDG synthesis at the very early germination stage is particularly important for chloroplast biogenesis. The MGD1 suppression down-regulated genes associated with the photorespiratory pathway in peroxisomes and mitochondria as well as those responsible for photosynthesis in chloroplasts and caused high expression of genes for the glyoxylate cycle. MGD1 function may link galactolipid synthesis with the coordinated transcriptional regulation of chloroplasts and other organelles during cotyledon greening.
Summary
The catalytic activity of mitogen‐activated protein kinases (MAPKs) is dynamically modified in plants. Since MAPKs have been shown to play important roles in a wide range of signaling ...pathways, the ability to monitor MAPK activity in living plant cells would be valuable. Here, we report the development of a genetically encoded MAPK activity sensor for use in Arabidopsis thaliana. The sensor is composed of yellow and blue fluorescent proteins, a phosphopeptide binding domain, a MAPK substrate domain and a flexible linker. Using in vitro testing, we demonstrated that phosphorylation causes an increase in the Förster resonance energy transfer (FRET) efficiency of the sensor. The FRET efficiency can therefore serve as a readout of kinase activity. We also produced transgenic Arabidopsis lines expressing this sensor of MAPK activity (SOMA) and performed live‐cell imaging experiments using detached cotyledons. Treatment with NaCl, the synthetic flagellin peptide flg22 and chitin all led to rapid gains in FRET efficiency. Control lines expressing a version of SOMA in which the phosphosite was mutated to an alanine did not show any substantial changes in FRET. We also expressed the sensor in a conditional loss‐of‐function double‐mutant line for the Arabidopsis MAPK genes MPK3 and MPK6. These experiments demonstrated that MPK3/6 are necessary for the NaCl‐induced FRET gain of the sensor, while other MAPKs are probably contributing to the chitin and flg22‐induced increases in FRET. Taken together, our results suggest that SOMA is able to dynamically report MAPK activity in living plant cells.
Significance Statement
The genetically encoded FRET sensor that we have developed should allow mitogen‐activated protein kinase (MAPK) signaling to be studied in living tissue with cellular resolution, which should lead to an improved understanding of the spatial and temporal dynamics of MAPK regulation in response to a range of different stimuli.
Key message
The disruption of the
SWL1
gene leads to a significant down regulation of chloroplast and secondary metabolites gene expression in
Arabidopsis thaliana
. And finally results in a ...dysfunction of chloroplast and plant growth.
Although the development of the chloroplast has been a consistent focus of research, the corresponding regulatory mechanisms remain unidentified. In this study, the CRISPR/Cas9 system was used to mutate the
SWL1
gene, resulting in albino cotyledons and variegated true leaf phenotype. Confocal microscopy and western blot of chloroplast protein fractions revealed that SWL1 localized in the chloroplast stroma. Electron microscopy indicated chloroplasts in the cotyledons of
swl1
lack well-defined grana and internal membrane structures, and similar structures have been detected in the albino region of variegated true leaves. Transcriptome analysis revealed that down regulation of chloroplast and nuclear gene expression related to chloroplast, including light harvesting complexes, porphyrin, chlorophyll metabolism and carbon metabolism in the
swl1
compared to wild-type plant. In addition, proteomic analysis combined with western blot analysis, showed that a significant decrease in chloroplast proteins of
swl1
. Furthermore, the expression of genes associated with secondary metabolites and growth hormones was also reduced, which may be attributed to SWL1 associated with absorption and fixation of inorganic carbon during chloroplast development. Together, the above findings provide valuable information to elucidate the exact function of SWL1 in chloroplast biogenesis and development.
Plants have developed numerous mechanisms to store hormones in inactive but readily available states, enabling rapid responses to environmental changes. The phytohormone auxin has a number of storage ...precursors, including indole-3-butyric acid (IBA), which is apparently shortened to active indole-3-acetic acid (IAA) in peroxisomes by a process similar to fatty acid β-oxidation. Whereas metabolism of auxin precursors is beginning to be understood, the biological significance of the various precursors is virtually unknown. We identified an Arabidopsis thaliana mutant that specifically restores IBA, but not IAA, responsiveness to auxin signaling mutants. This mutant is defective in PLEIOTROPIC DRUG RESISTANCE8 (PDR8)/PENETRATION3/ABCG36, a plasma membrane-localized ATP binding cassette transporter that has established roles in pathogen responses and cadmium transport. We found that pdr8 mutants display defects in efflux of the auxin precursor IBA and developmental defects in root hair and cotyledon expansion that reveal previously unknown roles for IBA-derived IAA in plant growth and development. Our results are consistent with the possibility that limiting accumulation of the IAA precursor IBA via PDR8-promoted efflux contributes to auxin homeostasis.
In plants, light represents an important environmental signal that triggers the production of photosynthetically active chloroplasts. This developmental switch is critical for plant survival because ...chlorophyll precursors that accumulate in darkness can be extremely destructive when illuminated. Thus, plants have evolved mechanisms to adaptively control plastid development during the transition into light. Here, we report that the gibberellin (GA)-regulated DELLA proteins play a crucial role in the formation of functional chloroplasts during deetiolation. We show that Arabidopsis thaliana DELLAs accumulating in etiolated cotyledons derepress chlorophyll and carotenoid biosynthetic pathways in the dark by repressing the transcriptional activity of the phytochrome-interacting factor proteins. Accordingly, dark-grown GA-deficient ga1-3 mutants (that accumulate DELLAs) display a similar gene expression pattern to wild-type seedlings grown in the light. Consistent with this, ga1-3 seedlings accumulate higher amounts of protochlorophyllide (a phototoxic chlorophyll precursor) in darkness but, surprisingly, are substantially more resistant to photooxidative damage following transfer into light. This is due to the DELLA-dependent upregulation of the photoprotective enzyme protochlorophyllide oxidoreductase (POR) in the dark. Our results emphasize the role of DELLAs in regulating the levels of POR, protochlorophyllide, and carotenoids in the dark and in protecting etiolated seedlings against photooxidative damage during initial light exposure.
Background and AimsDuring embryo development in most gymnosperms, the establishment of the shoot apical meristem (SAM) occurs concomitantly with the formation of a crown of cotyledons surrounding the ...SAM. It has previously been shown that the differentiation of cotyledons in somatic embryos of Picea abies is dependent on polar auxin transport (PAT). In the angiosperm model plant, Arabidopsis thaliana, the establishment of cotyledonary boundaries and the embryonal SAM is dependent on PAT and the expression of the CUP-SHAPED COTYLEDON (CUC) genes, which belong to the large NAC gene family. The aim of this study was to characterize CUC-like genes in a gymnosperm, and to elucidate their expression during SAM and cotyledon differentiation, and in response to PAT.MethodsSixteen Picea glauca NAC sequences were identified in GenBank and deployed to different clades within the NAC gene family using maximum parsimony analysis and Bayesian inference. Motifs conserved between angiosperms and gymnosperms were analysed using the motif discovery tool MEME. Expression profiles during embryo development were produced using quantitative real-time PCR. Protein conservation was analysed by introducing a P. abies CUC orthologue into the A. thaliana cuc1cuc2 double mutant.Key ResultsTwo full-length CUC-like cDNAs denoted PaNAC01 and PaNAC02 were cloned from P. abies. PaNAC01, but not PaNAC02, harbours previously characterized functional motifs in CUC1 and CUC2. The expression profile of PaNAC01 showed that the gene is PAT regulated and associated with SAM differentiation and cotyledon formation. Furthermore, PaNAC01 could functionally substitute for CUC2 in the A. thaliana cuc1cuc2 double mutant.ConclusionsThe results show that CUC-like genes with distinct signature motifs existed before the separation of angiosperms and gymnosperms approx. 300 million years ago, and suggest a conserved function between PaNAC01 and CUC1/CUC2.
Main conclusion
Tissue-specific occurrence and formation of endogenous sesquiterpene lactones has been assessed and suggests physiological function as antagonists of auxin-induced plant growth in ...sunflower.
Sunflower,
Helianthus annuus
, accumulate high concentrations of bioactive sesquiterpene lactones (STL) in glandular trichomes, but in addition, structurally different STL occur in only trace amounts in the inner tissues. The spatial and temporal production of these endogenous STL during early phases of plant development is widely unknown and their physiological function as putative natural growth regulators is yet speculative. By means of HPLC and MS analysis it was shown that costunolide, dehydrocostuslactone, 8-epixanthatin and tomentosin are already present in dry seeds and can be extracted in low amounts from cotyledons, hypocotyls and roots of seedlings during the first days after germination. Semi-quantitative and RT-qPCR experiments with genes of the key enzymes of two independent routes of the endogenous STL biosynthesis confirmed the early and individual expression in these organs and revealed a gradual down regulation during the first 72–96 h after germination. Light irradiation of the plants led to a fast, but transient increase of STL in parts of the hypocotyl which correlated with growth retardation of the stem. One-sided external application of costunolide on hypocotyls conferred reduced growth of the treated side, thus resulting in the curving of the stem towards the side of the application. This indicates the inhibiting effects of STL on plant growth. The putative function of endogenous STL in sunflower as antagonists of auxin in growth processes is discussed.
The period following seedling emergence is a particularly vulnerable stage in the plant life cycle. In Arabidopsis thaliana, the phytochrome-interacting factor (PIF) subgroup of ...basic-helix-loop-helix transcription factors has a pivotal role in regulating growth during this early phase, integrating environmental and hormonal signals. We previously showed that SPATULA (SPT), a PIF homolog, regulates seed dormancy. In this article, we establish that unlike PIFs, which mainly promote hypocotyl elongation, SPT is a potent regulator of cotyledon expansion. Here, SPT acts in an analogous manner to the gibberellin-dependent DELLAs, REPRESSOR OF GA1-3 and GIBBERELLIC ACID INSENSITIVE, which restrain cotyledon expansion alongside SPT. However, although DELLAs are not required for SPT action, we demonstrate that SPT is subject to negative regulation by DELLAs. Cross-regulation of SPT by DELLAs ensures that SPT protein levels are limited when DELLAs are abundant but rise following DELLA depletion. This regulation provides a means to prevent excessive growth suppression that would result from the dual activity of SPT and DELLAs, yet maintain growth restraint under DELLAdepleted conditions. We present evidence that SPT and DELLAs regulate common gene targets and illustrate that the balance of SPT and DELLA action depends on light quality signals in the natural environment.
Summary
Correct chloroplast development and function require co‐ordinated expression of chloroplast and nuclear genes. This is achieved through chloroplast signals that modulate nuclear gene ...expression in accordance with the chloroplast's needs. Genetic evidence indicates that GUN1, a chloroplast‐localized pentatricopeptide repeat (PPR) protein with a C‐terminal Small MutS‐Related (SMR) domain, is involved in integrating multiple developmental and stress‐related signals in both young seedlings and adult leaves. Recently, GUN1 was found to interact physically with factors involved in chloroplast protein homeostasis, and with enzymes of tetrapyrrole biosynthesis in adult leaves that function in various retrograde signalling pathways. Here we show that following perturbation of chloroplast protein homeostasis: (i) by growth in lincomycin‐containing medium; or (ii) in mutants defective in either the FtsH protease complex (ftsh), plastid ribosome activity (prps21‐1 and prpl11‐1) or plastid protein import and folding (cphsc70‐1), GUN1 influences NEP‐dependent transcript accumulation during cotyledon greening and also intervenes in chloroplast protein import.
Significance Statement
Correct chloroplast development and function require co‐ordinated regulation of chloroplast and nuclear gene expression. Here, we propose that GUN1, a main player of chloroplast‐to‐nucleus retrograde communication, influences NEP‐dependent transcript accumulation during cotyledon greening and also intervenes in chloroplast protein import.
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