Synthetic Strigolactone GR24 Improves Arabidopsis Somatic Embryogenesis through Changes in Auxin Responses

Somatic embryogenesis in Arabidopsis encompasses an induction phase requiring auxin as the inductive signal to promote cellular dedifferentiation and formation of the embryogenic tissue, and a developmental phase favoring the maturation of the embryos. Strigolactones (SLs) have been categorized as a novel group of plant hormones based on their ability to affect physiological phenomena in plants. The study analyzed the effects of synthetic strigolactone GR24, applied during the induction phase, on auxin response and formation of somatic embryos. The expression level of two SL biosynthetic genes, MOREAXILLARY GROWTH 3 and 4 (MAX3 and MAX4), which are responsible for the conversion of carotene to carotenal, increased during the induction phase of embryogenesis. Arabidopsis mutant studies indicated that the somatic embryo number was inhibited in max3 and max4 mutants, and this effect was reversed by applications of GR24, a synthetic strigolactone, and exacerbated by TIS108, a SL biosynthetic inhibitor. The transcriptional studies revealed that the regulation of GR24 and TIS108 on somatic embryogenesis correlated with changes in expression of AUXIN RESPONSIVE FACTORs 5, 8, 10, and 16, known to be required for the production of the embryogenic tissue, as well as the expression of WUSCHEL (WUS) and Somatic Embryogenesis Receptor-like Kinase 1 (SERK1), which are markers of cell dedifferentiation and embryogenic tissue formation. Collectively, this work demonstrated the novel role of SL in enhancing the embryogenic process in Arabidopsis and its requirement for inducing the expression of genes related to auxin signaling and production of embryogenic tissue.

A tonoplast-localized magnesium transporter is involved in stomatal opening in Arabidopsis

Plant stomata play an important role in CO2 uptake for photosynthesis and transpiration, but the mechanisms underlying stomatal opening and closing are still not completely understood. Here, through large-scale screening, we identified an Arabidopsis mutant (cst2 for closed stomata2) defective in stomatal opening under light condition. A map-based cloning combined with complementation test revealed that the mutant phenotype was caused by a nucleotide substitution of a gene, which domains show similarity to human Mg efflux transporter ACDP/CNNM. Functional analysis showed that CST2 encodes a tonoplast-localized transporter for Mg. This protein is constitutively and highly expressed in the guard cells. Furthermore, CST2 is phosphorylated by calcineurin B-like protein (CBL)-interacting protein kinases 26 (CIPK26) in vitro, which is probably required for its activation. Knockout of this gene resulted in stomatal closing and growth retardation under high Mg concentration conditions, while over-expression of this gene increased tolerance to high Mg. Our results indicate that CST2 plays an important role in maintaining Mg homeostasis in plant cells through sequestering Mg into vacuoles especially in guard cells and that this homeostasis is required for stomatal opening, which provide a novel insight into mechanism of stomatal opening in plants.

Uncoupling differential water usage from drought resistance in a dwarf Arabidopsis mutant

Understanding the molecular and physiological mechanisms of how plants respond to drought is paramount to breeding more drought resistant crops. Certain mutations or allelic variations result in plants with altered water-use requirements. To correctly identify genetic differences which confer a drought phenotype, plants with different genotypes must therefore be subjected to equal levels of drought stress. Many reports of advantageous mutations conferring drought resistance do not control for soil water content variations across genotypes and may therefore need to be re-examined. Here, we reassessed the drought phenotype of the Arabidopsis thaliana dwarf mutant, chiquita1-1 (also called cost1), by growing mutant seedlings together with the wild type to ensure uniform soil water availability across genotypes. Our results demonstrate that the dwarf phenotype conferred by loss of CHIQ1 function results in constitutively lower water usage, but not increased drought resistance.

A New Role for Plastid Thioredoxins in Seed Physiology in Relation to Hormone Regulation

In Arabidopsis seeds, ROS have been shown to be enabling actors of cellular signaling pathways promoting germination, but their accumulation under stress conditions or during aging leads to a decrease in the ability to germinate. Previous biochemical work revealed that a specific class of plastid thioredoxins (Trxs), the y-type Trxs, can fulfill antioxidant functions. Among the ten plastidial Trx isoforms identified in Arabidopsis, Trx y1 mRNA is the most abundant in dry seeds. We hypothesized that Trx y1 and Trx y2 would play an important role in seed physiology as antioxidants. Using reverse genetics, we found important changes in the corresponding Arabidopsis mutant seeds. They display remarkable traits such as increased longevity and higher and faster germination in conditions of reduced water availability or oxidative stress. These phenotypes suggest that Trxs y do not play an antioxidant role in seeds, as further evidenced by no changes in global ROS contents and protein redox status found in the corresponding mutant seeds. Instead, we provide evidence that marker genes of ABA and GAs pathways are perturbed in mutant seeds, together with their sensitivity to specific hormone inhibitors. Altogether, our results suggest that Trxs y function in Arabidopsis seeds is not linked to their previously identified antioxidant roles and reveal a new role for plastid Trxs linked to hormone regulation.

Phosphatidylglycerol synthesis facilitates plastid gene expression and light induction of nuclear photosynthetic genes

Phosphatidylglycerol (PG) is the only major phospholipid in the thylakoid membrane of chloroplasts. PG is essential for photosynthesis and loss of PG in Arabidopsis thaliana results in severe defects of growth and chloroplast development with decreased chlorophyll accumulation, impaired thylakoid formation, and downregulation of photosynthesis-associated genes encoded in nuclear and plastid genomes. However, how the absence of PG affects the gene expression and plant growth remains unclear. To elucidate this mechanism, we investigated the growth and transcriptional profiles of a PG-deficient Arabidopsis mutant pgp1-2 under various light conditions. Microarray analysis demonstrated that reactive oxygen species-responsive genes were upregulated in pgp1-2. Decreased growth light did not alleviated the impaired leaf development and the downregulation of photosynthesis-associated genes in pgp1-2, indicating limited impacts of photooxidative stress on the defects of pgp1-2. Illumination to dark-adapted pgp1-2 triggered downregulation of photosynthesis-associated nuclear-encoded genes (PhANGs), while plastid-encoded genes were constantly suppressed. Overexpression of GOLDEN2-LIKE1 (GLK1), a transcription factor regulating chloroplast development, in pgp1-2 upregulated PhANGs but not plastid-encoded genes along with chlorophyll accumulation. Our data suggest a broad impact of PG biosynthesis on nuclear-encoded genes partially via GLK1 and a specific involvement of this lipid in the plastid gene expression and plant development.

Altered metal distribution in the sr45-1 Arabidopsis mutant causes developmental defects

The plant SR (serine/arginine-rich) splicing factor SR45 plays important roles in several biological processes, such as splicing, DNA methylation, innate immunity, glucose regulation and ABA signaling. A homozygous Arabidopsis sr45-1 null mutant is viable, but exhibits diverse phenotypic alterations, including delayed root development, late flowering, shorter siliques with fewer seeds, narrower leaves and petals, and unusual numbers of floral organs. Here, we report that the sr45-1 mutant presents an unexpected constitutive iron deficiency phenotype characterized by altered metal distribution in the plant. RNA-Sequencing highlighted severe perturbations in metal homeostasis, phenylpropanoid pathway, oxidative stress responses, and reproductive development. Ionomic quantification and histochemical staining revealed strong iron accumulation in the sr45-1 root tissues accompanied by an iron starvation in aerial parts. We showed that some sr45-1 developmental abnormalities can be complemented by exogenous iron supply. Our findings provide new insight into the molecular mechanisms governing the phenotypes of the sr45-1 mutant.

A Hypomorphic Mutant of PHD Domain Protein Male Meiocytes Death 1

Meiosis drives reciprocal genetic exchanges and produces gametes with halved chromosome number, which is important for the genetic diversity, plant viability, and ploidy consistency of flowering plants. Alterations in chromosome dynamics and/or cytokinesis during meiosis may lead to meiotic restitution and the formation of unreduced microspores. In this study, we isolated an Arabidopsis mutant male meiotic restitution 1 (mmr1), which produces a small subpopulation of diploid or polyploid pollen grains. Cytological analysis revealed that mmr1 produces dyads, triads, and monads indicative of male meiotic restitution. Both homologous chromosomes and sister chromatids in mmr1 are separated normally, but chromosome condensation at metaphase I is slightly affected. The mmr1 mutant displayed incomplete meiotic cytokinesis. Supportively, immunostaining of the microtubular cytoskeleton showed that the spindle organization at anaphase II and mini-phragmoplast formation at telophase II are aberrant. The causative mutation in mmr1 was mapped to chromosome 1 at the chromatin regulator Male Meiocyte Death 1 (MMD1/DUET) locus. mmr1 contains a C-to-T transition at the third exon of MMD1/DUET at the genomic position 2168 bp from the start codon, which causes an amino acid change G618D that locates in the conserved PHD-finger domain of histone binding proteins. The F1 progenies of mmr1 crossing with knockout mmd1/duet mutant exhibited same meiotic defects and similar meiotic restitution rate as mmr1. Taken together, we here report a hypomorphic mmd1/duet allele that typically shows defects in microtubule organization and cytokinesis.

Divergence of three BRX homoeologs in Brassica rapa and its effect on leaf morphology

The leafy head characteristic is a special phenotype of Chinese cabbage resulting from artificial selection during domestication and breeding. BREVIS RADIX (BRX) has been suggested to control root elongation, shoot growth, and tiller angle in Arabidopsis and rice. In Brassica rapa, three BrBRX homoeologs have been identified, but only BrBRX.1 and BrBRX.2 were found to be under selection in leaf-heading accessions, indicating their functional diversification in leafy head formation. Here, we show that these three BrBRX genes belong to a plant-specific BRX gene family but that they have significantly diverged from other BRX-like members on the basis of different phylogenetic classifications, motif compositions and expression patterns. Moreover, although the expression of these three BrBRX genes differed, compared with BrBRX.3, BrBRX.1, and BrBRX.2 displayed similar expression patterns. Arabidopsis mutant complementation studies showed that only BrBRX.1 could rescue the brx root phenotype, whereas BrBRX.2 and BrBRX.3 could not. However, overexpression of each of the three BrBRX genes in Arabidopsis resulted in similar pleiotropic leaf phenotypes, including epinastic leaf morphology, with an increase in leaf number and leaf petiole length and a reduction in leaf angle. These leaf traits are associated with leafy head formation. Further testing of a SNP (T/C) in BrBRX.2 confirmed that this allele in the heading accessions was strongly associated with the leaf-heading trait of B. rapa. Our results revealed that all three BrBRX genes may be involved in the leaf-heading trait, but they may have functionally diverged on the basis of their differential expression.

Defects in Cell Wall Differentiation of the Arabidopsis Mutant rol1-2 Is Dependent on Cyclin-Dependent Kinase CDK8

Plant cells are encapsulated by cell walls whose properties largely determine cell growth. We have previously identified the rol1-2 mutant, which shows defects in seedling root and shoot development. rol1-2 is affected in the Rhamnose synthase 1 (RHM1) and shows alterations in the structures of Rhamnogalacturonan I (RG I) and RG II, two rhamnose-containing pectins. The data presented here shows that root tissue of the rol1-2 mutant fails to properly differentiate the cell wall in cell corners and accumulates excessive amounts of callose, both of which likely alter the physical properties of cells. A surr (suppressor of the rol1-2 root developmental defect) mutant was identified that alleviates the cell growth defects in rol1-2. The cell wall differentiation defect is re-established in the rol1-2 surr mutant and callose accumulation is reduced compared to rol1-2. The surr mutation is an allele of the cyclin-dependent kinase 8 (CDK8), which encodes a component of the mediator complex that influences processes central to plant growth and development. Together, the identification of the surr mutant suggests that changes in cell wall composition and turnover in the rol1-2 mutant have a significant impact on cell growth and reveals a function of CDK8 in cell wall architecture and composition.