D14 Mutants Research Articles

AtMYBS1 negatively regulates heat tolerance by directly repressing the expression of MAX1 required for strigolactone biosynthesis in Arabidopsis

Heat stress caused by global warming requires the development of thermotolerant crops to sustain yield. It is necessary to understand the molecular mechanisms that underlie heat tolerance in plants. Strigolactones (SLs) are a class of carotenoid-derived phytohormones that regulate plant development and responses to abiotic or biotic stresses. Although SL biosynthesis and signaling processes are well established, genes that directly regulate SL biosynthesis have rarely been reported. Here, we report that the MYB-like transcription factor AtMYBS1/AtMYBL, whose gene expression is repressed by heat stress, functions as a negative regulator of heat tolerance by directly inhibiting SL biosynthesis in Arabidopsis. Overexpression of AtMYBS1 led to heat hypersensitivity, whereas atmybs1 mutants displayed increased heat tolerance. Expression of MAX1, a critical enzyme in SL biosynthesis, was induced by heat stress and downregulated in AtMYBS1-overexpression (OE) plants but upregulated in atmybs1 mutants. Overexpression of MAX1 in the AtMYBS1-OE background reversed the heat hypersensitivity of AtMYBS1-OE plants. Loss of MAX1 function in the atmyb1 background reversed the heat-tolerant phenotypes of atmyb1 mutants. Yeast one-hybrid assays, chromatin immunoprecipitation‒qPCR, and transgenic analyses demonstrated that AtMYBS1 directly represses MAX1 expression through the MYB binding site in the MAX1 promoter in vivo. The atmybs1d14 double mutant, like d14 mutants, exhibited hypersensitivity to heat stress, indicating the necessary role of SL signaling in AtMYBS1-regulated heat tolerance. Our findings provide new insights into the regulatory network of SL biosynthesis, facilitating the breeding of heat-tolerant crops to improve crop production in a warming world.

Quantitative proteomics implicates YggT in streptomycin resistance in Salmonella enterica serovar Enteritidis.

To identify proteins that may be associated with antibiotic resistance in the multidrug-resistant Salmonella enterica D14, by constructing proteomic profiles using mass spectrometry-based label-free quantitative proteomics (LFQP). D14 was cultured with four antibiotics (ampicillin, nalidixic acid, streptomycin, and tetracycline) separately. Subsequently, the findings from an equal combination of the four cultures were compared with the profile of sensitive S. enterica 104. 2255 proteins, including 149 differentially up-regulated proteins, were identified. Many of these up-regulated proteins were associated with flagellar assembly and chemotaxis, two-component system, amino acid metabolism, β-lactam resistance, and transmembrane transport. A subset of 10 genes was evaluated via quantitative real-time PCR (qPCR), followed by the construction of cheR, fliS, fliA, arnA, and yggT deletion mutants. Only the yggT-deleted D14 mutant showed decrease in streptomycin resistance, whereas the other deletions had no effect. Furthermore, complementation of yggT and the overexpression of yggT in S. enterica ATCC 14028 increased the streptomycin resistance. Additionally, spot dilution assay results confirmed that Salmonella strains, harboring yggT, exhibited an advantage in the presence of streptomycin. The above proteomic and mutagenic analyses revealed that yggT is involved in streptomycin resistance in S. enterica.

Differential role of MAX2 and strigolactones in pathogen, ozone, and stomatal responses.

Strigolactones are a group of phytohormones that control developmental processes including shoot branching and various plant–environment interactions in plants. We previously showed that the strigolactone perception mutant more axillary branches 2 (max2) has increased susceptibility to plant pathogenic bacteria. Here we show that both strigolactone biosynthesis (max3 and max4) and perception mutants (max2 and dwarf14) are significantly more sensitive to Pseudomonas syringae DC3000. Moreover, in response to P. syringae infection, high levels of SA accumulated in max2 and this mutant was ozone sensitive. Further analysis of gene expression revealed no major role for strigolactone in regulation of defense gene expression. In contrast, guard cell function was clearly impaired in max2 and depending on the assay used, also in max3, max4, and d14 mutants. We analyzed stomatal responses to stimuli that cause stomatal closure. While the response to abscisic acid (ABA) was not impaired in any of the mutants, the response to darkness and high CO2 was impaired in max2 and d14‐1 mutants, and to CO2 also in strigolactone synthesis (max3, max4) mutants. To position the role of MAX2 in the guard cell signaling network, max2 was crossed with mutants defective in ABA biosynthesis or signaling. This revealed that MAX2 acts in a signaling pathway that functions in parallel to the guard cell ABA signaling pathway. We propose that the impaired defense responses of max2 are related to higher stomatal conductance that allows increased entry of bacteria or air pollutants like ozone. Furthermore, as MAX2 appears to act in a specific branch of guard cell signaling (related to CO2 signaling), this protein could be one of the components that allow guard cells to distinguish between different environmental conditions.

Strigolactones positively regulate defense against Magnaporthe oryzae in rice (Oryza sativa)

This study presents evidence that strigolactones (SLs) promote defense against devastating rice blast fungal pathogen Magnaporthe oryzae. Impairment in either SL-biosynthetic dwarf17 (d17) or –signaling (d14) led to increased susceptibility towards M. oryzae. Comparative transcriptome profiling of the SL-signaling d14 mutant and WT plants revealed that a large number of defense-associated genes including hydrogen peroxide (H2O2)-, ethylene- and cell wall-synthesis-related genes were remarkably suppressed in d14 with respect to that of WT plants, during M. oryzae infection. In addition, various KEGG metabolic pathways related to sugar metabolism were significantly suppressed in the d14 plants compared to WT, during M. oryzae infection. Accordingly, WT plants accumulated increased levels of H2O2 and soluble sugar content compared to that of d17 and d14 in response to M. oryzae infection. Altogether, these results propose that SLs positively regulated rice defense against M. oryzae through involvement in the induction of various defense associated genes/pathways.

Strigolactones shape the rhizomicrobiome in rice (Oryza sativa)

The rhizomicrobiome helps the host plant to better adapt to environmental stresses. In contrast, plant-derived metabolic substances, including phytohormones, play an active role in structuring rhizomicrobiome. Although strigolactones (SLs), a group of phytohormones, serve as potential rhizosphere signaling molecules, their contributions in shaping the rice (Oryza sativa) rhizomicrobiome remain elusive. To address this issue, we compared the rhizomicrobiome of rice mutants defective in either SL biosynthesis or signaling and wild-type (WT) plants. To understand whether SL-regulated metabolic pathways shape the rhizomicrobiome, a correlation network analysis was conducted among the metabolic pathway-related genes and the rhizomicrobiome of rice. Compared to WT, higher bacterial richness (evidenced by the operational taxonomic unit richness) and lower fungal diversity (evidenced by the Shannon index) were observed in both SL deficient dwarf17 (d17) and signaling (d14) mutants. Additionally, remarkable differences were observed in the composition of a large number of bacterial communities than the fungal communities in the d17 and d14 mutants with respect to the WT. The abundance of certain beneficial bacterial taxa, including Nitrosomonadaceae and Rhodanobacter, were significantly decreased in both mutants relative to the WT. Correlation network analysis between SL-regulated metabolic pathway-associated genes and rhizomicrobiome proposed a role for SL-dependent metabolic pathways in shaping rhizomicrobiome composition. Taken together, our study suggests that SL biosynthesis and signaling play a key role in determining the rice rhizomicrobiome, directly or indirectly, through the mediation of distinct metabolic pathways. Based on our findings, the genetic modulation of rice SL biosynthesis and/or signaling pathways may help to recruit/increase the abundance of the desired rhizomicrobiome, which may assist in the stress resilience of rice.

Rice DWARF14 acts as an unconventional hormone receptor for strigolactone.

Strigolactones (SLs) act as an important class of phytohormones to regulate plant shoot branching, and also serve as rhizosphere signals to mediate interactions of host plants with soil microbes and parasitic weeds. SL receptors in dicots, such as DWARF14 in Arabidopsis (AtD14), RMS3 in pea, and ShHTL7 in Striga, serve as unconventional receptors that hydrolyze SLs into a D-ring-derived intermediate CLIM and irreversibly bind CLIM to trigger SL signal transduction. Here, we show that D14 from the monocot rice can complement Arabidopsis d14 mutant and interact with the SL signaling components in Arabidopsis. Our results further reveal that rice D14, similar to SL receptors in dicots, also serves as an unconventional hormone receptor that generates and irreversibly binds the active form of SLs. These findings uncover the conserved functions of D14 proteins in monocots and dicots.

Characterization of DWARF14 Genes in Populus.

Strigolactones are a new class of plant hormones regulating shoot branching and symbiotic interactions with arbuscular mycorrhizal fungi. Studies of branching mutants in herbaceous plants have identified several key genes involved in strigolactone biosynthesis or signaling. The strigolactone signal is perceived by a member of the α/β-fold hydrolase superfamily, known as DWARF14 (D14). However, little is known about D14 genes in the woody perennial plants. Here we report the identification of D14 homologs in the model woody plant Populus trichocarpa. We showed that there are two D14 homologs in P. trichocarpa, designated as PtD14a and PtD14b that are over 95% similar at the amino acid level. Expression analysis indicated that the transcript level of PtD14a is generally more abundant than that of PtD14b. However, only PtD14a was able to complement Arabidopsis d14 mutants, suggesting that PtD14a is the functional D14 ortholog. Amino acid alignment and structural modeling revealed substitutions of several highly conserved amino acids in the PtD14b protein including a phenylalanine near the catalytic triad of D14 proteins. This study lays a foundation for further characterization of strigolactone pathway and its functions in the woody perennial plants.

The D3 F‐box protein is a key component in host strigolactone responses essential for arbuscular mycorrhizal symbiosis

Arbuscular mycorrhiza (AM) represents an ancient endosymbiosis between plant roots and Glomeromycota fungi. Strigolactones (SLs), plant-derived terpenoid lactones, activate hyphal branching of AM fungi before physical contact. Lack of SL biosynthesis results in lower colonization of AM fungi. The F-box protein, DWARF3 (D3), and the hydrolase family protein DWARF14 (D14) are crucial for SL responses in rice. Here we conducted AM fungal colonization assays with the SL-insensitive d3 and d14 mutants. The d3 mutant exhibited strong defects in AM fungal colonization, whereas the d14 mutant showed higher AM fungal colonization. As D14 has a homologous protein, D14-LIKE, we generated D14-LIKE knockdown lines by RNA interference in the wildtype and d14 background. D14 and D14-LIKE double knockdown lines exhibited similar colonization rates as those of the d14-1 mutant. D3 is crucial for establishing AM symbiosis in rice, whereas D14 and D14-LIKE are not. Our results suggest distinct roles for these SL-related components in AM symbiosis.

D14, a Strigolactone-Insensitive Mutant of Rice, Shows an Accelerated Outgrowth of Tillers

Recent studies using highly branched mutants of pea, Arabidopsis and rice have demonstrated that strigolactones, a group of terpenoid lactones, act as a new hormone class, or its biosynthetic precursors, in inhibiting shoot branching. Here, we provide evidence that DWARF14 (D14) inhibits rice tillering and may act as a new compo-nent of the strigolactone-dependent branching inhibition pathway. The d14 mutant exhibits increased shoot branch-ing with reduced plant height like the previously characterized strigolactone-deficient and -insensitive mutants d10 and d3, respectively. The d10-1 d14-1 double mutant is phenotypically indistinguishable from the d10-1 and d14-1 single mutants, consistent with the idea that D10 and D14 function in the same pathway. However, unlike with d10, the d14 branching phenotype could not be rescued by exogenous strigolactones. In addition, the d14 mutant contained a higher level of 2'-epi-5-deoxystrigol than the wild type. Positional cloning revealed that D14 encodes a protein of the alpha/beta-fold hydrolase superfamily, some members of which play a role in metabolism or signaling of plant hormones. We propose that D14 functions downstream of strigolactone synthesis, as a component of hormone signaling or as an enzyme that participates in the conversion of strigolactones to the bioactive form.

Study of novel d1 alleles, defective mutants of the .ALPHA. subunit of heterotrimeric G-protein in rice

It has been shown that the disruption of the alpha-subunit gene of heterotorimeric G-proteins (Galpha) results in dwarf traits, the erection of leaves and the setting of small seeds in rice. These mutants are called d1. We have studied the expression profiles of the transcripts and translation products of rice Galpha in ten alleles of d1 including five additional alleles newly identified. By RT-PCR, the transcripts of the Galpha gene were detected in the all d1 alleles. By western blot, the Galpha proteins were not detected in the plasma membrane fractions of the d1 alleles with the exception of d1-4. In d1-4, one amino acid change in the GTP-binding box A of the Galpha protein was occurred and even in this case the Galpha protein was only just detectable in the plasma membrane fraction. Given that the Galpha protein did not accumulate in the plasma membrane fraction in d1-8 which has a deletion of just a single amino acid in the Galpha protein, it is likely that a proper conformation of the Galpha is necessary for accumulation of Galpha protein in the plasma membrane. Nine alleles of d1 showed a severer phenotype whilst d1-4 exhibited a mild phenotype with respect to seed size and elongation pattern of internodes. As brassinosteroid signaling was known to be partially impaired in d1s, the sensitivity to 24-epibrassinolide (24-epiBL) was compared among d1 alleles in a T65 genetic background. Only d1-4 showed responses similar to wild type rice. The results show that the d1-4 mutant is a mild allele in terms of the phenotype and mild hyposensitivity to the exogenously applied 24-epiBL.