The CRISPR/Cas9-Mediated Modulation of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 in Alfalfa Leads to Distinct Phenotypic Outcomes

Alfalfa (Medicago sativa L.) is the most widely grown perennial leguminous forage and is an essential component of the livestock industry. Previously, the RNAi-mediated down-regulation of alfalfa SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 (MsSPL8) was found to lead to increased branching, regrowth and biomass, as well as enhanced drought tolerance. In this study, we aimed to further characterize the function of MsSPL8 in alfalfa using CRISPR/Cas9-induced mutations in this gene. We successfully generated alfalfa genotypes with small insertions/deletions (indels) at the target site in up to three of four MsSPL8 alleles in the first generation. The efficiency of editing appeared to be tightly linked to the particular gRNA used. The resulting genotypes displayed consistent morphological alterations, even with the presence of up to two wild-type MsSPL8 alleles, including reduced leaf size and early flowering. Other phenotypic effects appeared to be dependent upon mutational dosage, with those plants with the highest number of mutated MsSPL8 alleles also exhibiting significant decreases in internode length, plant height, shoot and root biomass, and root length. Furthermore, MsSPL8 mutants displayed improvements in their ability to withstand water-deficit compared to empty vector control genotypes. Taken together, our findings suggest that allelic mutational dosage can elicit phenotypic gradients in alfalfa, and discrepancies may exist in terms of MsSPL8 function between alfalfa genotypes, growth conditions, or specific alleles. In addition, our results provide the foundation for further research exploring drought tolerance mechanisms in a forage crop.

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Do Arabidopsis Squamosa promoter binding Protein-Like genes act together in plant acclimation to copper or zinc deficiency?

10.1101/590182 ◽

2019 ◽

Author(s):

Anna Schulten ◽

Lucas Bytomski ◽

Julia Quintana ◽

Maria Bernal ◽

Ute Kraemer

Keyword(s):

Binding Protein ◽

Growth Conditions ◽

Zn Deficiency ◽

Gene Promoters ◽

Wild Type ◽

Triple Mutant ◽

Protein Activation ◽

Cu Deficiency ◽

Squamosa Promoter Binding Protein ◽

Genes Encoding

The genome of Arabidopsis thaliana encodes approximately 260 copper (Cu)-dependent proteins, which include enzymes in central pathways of photosynthesis, respiration and responses to environmental stress. Under Cu-deficient growth conditions, Squamosa promoter binding Protein-Like 7 (SPL7) activates the transcription of genes encoding Cu acquisition systems and mediates a metabolic reorganization to economize on Cu. The transcription factor SPL7 groups among comparably large proteins in the SPL family, which additionally comprises a second group of small SPL proteins targeted by miRNA156 with roles in plant development. SPL7 shares extended regions of sequence homology with SPL1 and SPL12. Therefore, we investigated the possibility of a functional overlap between these three members of the group of large SPL family proteins. We compared the spl1 spl12 double mutant and the spl1 spl7 spl12 triple mutant with the wild type and the spl7 single mutant under normal and Cu-deficient growth conditions. Biomass production, chlorophyll content and tissue elemental composition at the seedling stage, as well as plant and flower morphology during reproductive stages, confirmed the involvement of SPL7, but provided no indication for important roles of SPL1 or SPL12, in the acclimation of Arabidopsis to Cu deficiency. Furthermore, we analyzed the effects of zinc (Zn) deficiency on the same set of mutants. Different from what is known in the green alga Chlamydomonas reinhardtii, Arabidopsis did not activate Cu deficiency responses under Zn deficiency, and there was no Cu overaccumulation in either shoot or root tissues of Zn-deficient wild-type plants. Known Zn deficiency responses were unaltered in spl7, spl1 spl12 and spl1 spl7 spl12 mutants. We observed that CuZnSOD activity is strongly downregulated in Zn-deficient A. thaliana, in association with an about 94% reduction in the abundance of the CDS2 transcript, a known target of miR398. However, different from the known Cu deficiency responses of Arabidopsis, this Zn deficiency response was independent of SPL7 and not associated with an upregulation of MIR398b primary transcript levels. Our data suggest that there is no conservation in A. thaliana of the crosstalk between Zn and Cu homeostasis mediated by the single SPL family protein CRR1 in Chlamydomonas. In the future, resolving how the specificity of SPL protein activation and recognition of target gene promoters is achieved will advance our understanding of the specific functions of different SPL family proteins in either Cu deficiency response regulation or growth and development of land plants.

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The Arabidopsis selenium-binding protein confers tolerance to toxic levels of selenium

Functional Plant Biology ◽

10.1071/fp05090 ◽

2005 ◽

Vol 32 (10) ◽

pp. 881 ◽

Cited By ~ 31

Author(s):

Adamantia Agalou ◽

Andreas Roussis ◽

Herman P. Spaink

Keyword(s):

Transgenic Plants ◽

Gene Silencing ◽

Binding Protein ◽

Normal Growth ◽

Growth Conditions ◽

Growth Responses ◽

Wild Type ◽

Selenium Toxicity ◽

Transcript Levels ◽

Expression Levels

In the Arabidopsis genome there are three highly conserved homologues of the mammalian 56-kD selenium-binding protein (SBP). To study the function of SBP in this model plant, we used a transgenic approach by constitutively overexpressing and down-regulating the endogenous Atsbp1 gene. In the latter case, we employed both a conventional antisense method and gene silencing by intron-containing hairpin RNAs. Atsbp1-overexpressing and silenced plants were phenotypically normal, under standard growth conditions, when compared with wild type plants. Transgenic plants exhibited different growth responses to exogenously supplied selenite, which correlated with the expression levels of Atsbp1. Plants with increased Atsbp1 transcript levels showed enhanced tolerance to selenite, while plants with reduced levels were more sensitive. Our results indicate that, although Atsbp1 does not play a detectable role in the regulation of developmental processes under normal growth conditions, it appears to be involved in processes controlling tolerance of Arabidopsis to selenium toxicity.

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The regulation of post-germinative transition from the cotyledon- to vegetative-leaf stages by microRNA-targeted SQUAMOSA PROMOTER-BINDING PROTEIN LIKE13 in Arabidopsis

Seed Science Research ◽

10.1017/s0960258510000073 ◽

2010 ◽

Vol 20 (2) ◽

pp. 89-96 ◽

Cited By ~ 40

Author(s):

Ruth C. Martin ◽

Masashi Asahina ◽

Po-Pu Liu ◽

Jessica R. Kristof ◽

Jennifer L. Coppersmith ◽

...

Keyword(s):

Binding Protein ◽

Leaf Primordium ◽

Mutant Form ◽

Wild Type ◽

Cotyledon Stage ◽

Squamosa Promoter Binding Protein ◽

Early Seedling ◽

Early Seedling Development ◽

Vegetative Leaf

AbstractGermination and early seedling development are critical for successful stand establishment of plants. Following germination, the cotyledons, which are derived from embryonic tissue, emerge from the seed. Arabidopsis seedlings at post-germinative stages are supported mainly by the supply of nutrition from the cotyledons until vegetative leaves emerge and initiate photosynthesis. The switch to autotrophic growth is a significant transition at the post-germinative stage. Here, we provide evidence that down-regulation of SQUAMOSA PROMOTER-BINDING PROTEIN LIKE13 (SPL13) by microRNA156 (miR156) plays an important role in the regulation of the post-germinative switch from the cotyledon stage to the vegetative-leaf stage. Silent mutations created in the SPL13 sequence in the region that is complementary to the miR156 sequence caused the deregulation of the mutant form of SPL13 (mSPL13) mRNA from miR156. Mutant seedlings over-accumulated miRNA-resistant messages and exhibited a delay in the emergence of vegetative leaves compared to wild-type seedlings. The delay was not observed in control transgenic plants expressing non-mutated SPL13, indicating that the phenotype was caused specifically by the silent mutations and deregulation of SPL13 from miR156. Characterization of the SPL13 promoter indicated that this gene is expressed mainly in the hypocotyl and affects leaf primordium development. These results suggest that the repression of SPL13 by miR156 is essential for normal post-germinative growth in Arabidopsis.

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Characterization of the Role of SPL9 in Drought Stress Tolerance in Medicago sativa

International Journal of Molecular Sciences ◽

10.3390/ijms21176003 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6003

Author(s):

Alexandria Hanly ◽

Jim Karagiannis ◽

Qing Shi Mimmie Lu ◽

Lining Tian ◽

Abdelali Hannoufa

Keyword(s):

Drought Tolerance ◽

Medicago Sativa ◽

Anthocyanin Biosynthesis ◽

Wild Type ◽

Yield And Quality ◽

Squamosa Promoter Binding Protein ◽

Drought Conditions ◽

Relative Water

Extreme environmental conditions, such as drought, are expected to increase in frequency and severity due to climate change, leading to substantial deficiencies in crop yield and quality. Medicago sativa (alfalfa) is an important crop that is relied upon as a staple source of forage in ruminant feed. Despite its economic importance, alfalfa production is constrained by abiotic stress, including drought. In this report, we investigate the role of Squamosa Promoter Binding Protein-Like 9 (SPL9), a target of miR156, in drought tolerance. Transgenic alfalfa plants with RNAi-silenced MsSPL9 (SPL9-RNAi) were compared to wild-type (WT) alfalfa for phenotypic changes and drought tolerance indicators. In SPL9-RNAi plants, both stem thickness and plant height were reduced in two- and six-month-old alfalfa, respectively; however, yield was unaffected. SPL9-RNAi plants showed less leaf senescence and had augmented relative water content under drought conditions, indicating that SPL9-RNAi plants had greater drought tolerance potential than WT plants. Interestingly, SPL9-RNAi plants accumulated more stress-alleviating anthocyanin compared to WT under both drought and well-watered control conditions, suggesting that MsSPL9 may contribute to drought tolerance in alfalfa, at least in part, by regulating anthocyanin biosynthesis. The results suggest that targeting MsSPL9 is a suitable means for improving alfalfa resilience towards drought conditions.

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The Lipid Lysyl-Phosphatidylglycerol Is Present in Membranes of Rhizobium tropici CIAT899 and Confers Increased Resistance to Polymyxin B Under Acidic Growth Conditions

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-20-11-1421 ◽

2007 ◽

Vol 20 (11) ◽

pp. 1421-1430 ◽

Cited By ~ 65

Author(s):

Christian Sohlenkamp ◽

Kanaan A. Galindo-Lagunas ◽

Ziqiang Guan ◽

Pablo Vinuesa ◽

Sally Robinson ◽

...

Keyword(s):

Sinorhizobium Meliloti ◽

Minimal Medium ◽

Polymyxin B ◽

Growth Conditions ◽

Membrane Lipid ◽

Low Ph ◽

Wild Type ◽

Rhizobium Tropici ◽

Gram Negative ◽

Inducible Genes

Lysyl-phosphatidylglycerol (LPG) is a well-known membrane lipid in several gram-positive bacteria but is almost unheard of in gram-negative bacteria. In Staphylococcus aureus, the gene product of mprF is responsible for LPG formation. Low pH-inducible genes, termed lpiA, have been identified in the gram-negative α-proteobacteria Rhizobium tropici and Sinorhizobium medicae in screens for acid-sensitive mutants and they encode homologs of MprF. An analysis of the sequenced bacterial genomes reveals that genes coding for homologs of MprF from S. aureus are present in several classes of organisms throughout the bacterial kingdom. In this study, we show that the expression of lpiA from R. tropici in the heterologous hosts Escherichia coli and Sinorhizobium meliloti causes formation of LPG. A wild-type strain of R. tropici forms LPG (about 1% of the total lipids) when the cells are grown in minimal medium at pH 4.5 but not when grown in minimal medium at neutral pH or in complex tryptone yeast (TY) medium at either pH. LPG biosynthesis does not occur when lpiA is deleted and is restored upon complementation of lpiA-deficient mutants with a functional copy of the lpiA gene. When grown in the low-pH medium, lpiA-deficient rhizobial mutants are over four times more susceptible to the cationic peptide polymyxin B than the wild type.

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The DNA Binding Protein Rfg1 Is a Repressor of Filamentation inCandida albicans

Genetics ◽

10.1093/genetics/157.4.1503 ◽

2001 ◽

Vol 157 (4) ◽

pp. 1503-1512 ◽

Cited By ~ 1

Author(s):

Roy A Khalaf ◽

Richard S Zitomer

Keyword(s):

Dna Binding ◽

Binding Protein ◽

Hyphal Growth ◽

Dna Binding Protein ◽

Homozygous Deletion ◽

Wild Type ◽

Pathogenic Yeast ◽

Repression Complex ◽

Type Gene ◽

Repression Domain

AbstractWe have identified a repressor of hyphal growth in the pathogenic yeast Candida albicans. The gene was originally cloned in an attempt to characterize the homologue of the Saccharomyces cerevisiae Rox1, a repressor of hypoxic genes. Rox1 is an HMG-domain, DNA binding protein with a repression domain that recruits the Tup1/Ssn6 general repression complex to achieve repression. The C. albicans clone also encoded an HMG protein that was capable of repression of a hypoxic gene in a S. cerevisiae rox1 deletion strain. Gel retardation experiments using the purified HMG domain of this protein demonstrated that it was capable of binding specifically to a S. cerevisiae hypoxic operator DNA sequence. These data seemed to indicate that this gene encoded a hypoxic repressor. However, surprisingly, when a homozygous deletion was generated in C. albicans, the cells became constitutive for hyphal growth. This phenotype was rescued by the reintroduction of the wild-type gene on a plasmid, proving that the hyphal growth phenotype was due to the deletion and not a secondary mutation. Furthermore, oxygen repression of the hypoxic HEM13 gene was not affected by the deletion nor was this putative ROX1 gene regulated positively by oxygen as is the case for the S. cerevisiae gene. All these data indicate that this gene, now designated RFG1 for Repressor of Filamentous Growth, is a repressor of genes required for hyphal growth and not a hypoxic repressor.

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Overexpression of the pitaya phosphoethanolamine N-methyltransferase gene (HpPEAMT) enhanced simulated drought stress in tobacco

Plant Cell Tissue and Organ Culture (PCTOC) ◽

10.1007/s11240-021-02040-3 ◽

2021 ◽

Author(s):

Ai-Hua Wang ◽

Lan Yang ◽

Xin-Zhuan Yao ◽

Xiao-Peng Wen

Keyword(s):

Drought Stress ◽

Stress Response ◽

Transgenic Plants ◽

Drought Tolerance ◽

Transgenic Tobacco ◽

Sequence Similarity ◽

Amino Acid Sequence Similarity ◽

Transgenic Tobacco Plants ◽

Wild Type ◽

Tobacco Plants

AbstractPhosphoethanolamine N-methyltransferase (PEAMTase) catalyzes the methylation of phosphoethanolamine to produce phosphocholine and plays an important role in the abiotic stress response. Although the PEAMT genes has been isolated from many species other than pitaya, its role in the drought stress response has not yet been fully elucidated. In the present study, we isolated a 1485 bp cDNA fragment of HpPEAMT from pitaya (Hylocereus polyrhizus). Phylogenetic analysis showed that, during its evolution, HpPEAMT has shown a high degree of amino acid sequence similarity with the orthologous genes in Chenopodiaceae species. To further investigate the function of HpPEAMT, we generated transgenic tobacco plants overexpressing HpPEAMT, and the transgenic plants accumulated significantly more glycine betaine (GB) than did the wild type (WT). Drought tolerance trials indicated that, compared with those of the wild-type (WT) plants, the roots of the transgenic plants showed higher drought tolerance ability and exhibited improved drought tolerance. Further analysis revealed that overexpression of HpPEAM in Nicotiana tabacum resulted in upregulation of transcript levels of GB biosynthesis-related genes (NiBADH, NiCMO and NiSDC) in the leaves. Furthermore, compared with the wild-type plants, the transgenic tobacco plants displayed a significantly lower malondialdehyde (MDA) accumulation and higher activities of the superoxide dismutase (SOD) and peroxidase (POD) antioxidant enzymes under drought stress. Taken together, our results suggested that HpPEAMT enhanced the drought tolerance of transgenic tobacco.

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Isolation of a gene required for programmed initiation of development by Aspergillus nidulans.

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3827 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3827-3833 ◽

Cited By ~ 41

Author(s):

T H Adams ◽

W A Hide ◽

L N Yager ◽

B N Lee

Keyword(s):

Life Cycle ◽

Aspergillus Nidulans ◽

Optimal Growth ◽

Growth Conditions ◽

Nutrient Deprivation ◽

Deletion Mutants ◽

Wild Type ◽

Microbial Development ◽

Type Strains ◽

Posttranscriptional Level

In contrast to many other cases in microbial development, Aspergillus nidulans conidiophore production initiates primarily as a programmed part of the life cycle rather than as a response to nutrient deprivation. Mutations in the acoD locus result in "fluffy" colonies that appear to grow faster than the wild type and proliferate as undifferentiated masses of vegetative cells. We show that unlike wild-type strains, acoD deletion mutants are unable to make conidiophores under optimal growth conditions but can be induced to conidiate when growth is nutritionally limited. The requirement for acoD in conidiophore development occurs prior to activation of brlA, a primary regulator of development. The acoD transcript is present both in vegetative hyphae prior to developmental induction and in developing cultures. However, the effects of acoD mutations are detectable only after developmental induction. We propose that acoD activity is primarily controlled at the posttranscriptional level and that it is required to direct developmentally specific changes that bring about growth inhibition and activation of brlA expression to result in conidiophore development.

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Genome-Wide Analysis of Poplar SQUAMOSA-Promoter-Binding Protein (SBP) Family under Salt Stress

Forests ◽

10.3390/f12040413 ◽

2021 ◽

Vol 12 (4) ◽

pp. 413

Author(s):

Qing Guo ◽

Li Li ◽

Kai Zhao ◽

Wenjing Yao ◽

Zihan Cheng ◽

...

Keyword(s):

Salt Stress ◽

Abiotic Stress ◽

Stress Responses ◽

Growth And Development ◽

Binding Protein ◽

Gene Segment ◽

Gene Expression Pattern ◽

Genome Wide Analysis ◽

Genome Wide ◽

Squamosa Promoter Binding Protein

SQUAMOSA promoter binding protein (SBP) is a kind of plant-specific transcription factor, which plays a crucial role in stress responses and plant growth and development by activating and inhibiting the transcription of multiple target genes. In this study, a total of 30 SBP genes were identified from Populus trichocarpa genome and randomly distributed on 16 chromosomes in poplar. According to phylogenetic analysis, the PtSBPs can be divided into six categories, and 14 out of the genes belong to VI. Furthermore, the SBP genes in VI were proved to have a targeting relationship with miR156. The homeopathic element analysis showed that the promoters of poplar SBP genes mainly contain the elements involved in growth and development, abiotic stress and hormone response. In addition, there existed 10 gene segment duplication events in the SBP gene duplication analysis. Furthermore, there were four poplar and Arabidopsis orthologous gene pairs among the poplar SBP members. What is more, poplar SBP gene family has diverse gene expression pattern under salt stress. As many as nine SBP members were responding to high salt stress and six members possibly participated in growth development and abiotic stress. Yeast two-hybrid experiments indicated that PtSBPs can form heterodimers to interact in the transcriptional regulatory networks. The genome-wide analysis of poplar SBP family will contribute to function characterization of SBP genes in woody plants.

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