The AtMYB60 transcription factor regulates stomatal opening by modulating oxylipin synthesis in guard cells

Stomata are epidermal pores formed by pairs of specialized guard cells, which regulate gas exchanges between the plant and the atmosphere. Modulation of transcription has emerged as an important level of regulation of stomatal activity. The AtMYB60 transcription factor was previously identified as a positive regulator of stomatal opening, although the details of its function remain unknown. Here, we propose a role for AtMYB60 as a negative modulator of oxylipins synthesis in stomata. The atmyb60-1 mutant shows reduced stomatal opening and accumulates increased levels of 12-oxo-phytodienoic acid (12-OPDA), jasmonic acid (JA) and jasmonoyl-l-isoleucine (JA-Ile) in guard cells. We provide evidence that 12-OPDA triggers stomatal closure independently of JA and cooperatively with abscisic acid (ABA) in atmyb60-1. Our study highlights the relevance of oxylipins metabolism in stomatal regulation and indicates AtMYB60 as transcriptional integrator of ABA and oxylipins responses in guard cells.

Transcriptional regulation of flavonoid biosynthesis in Artemisia annua by AaYABBY5

Artemisia annua is a medicinal plant rich in terpenes and flavonoids with useful biological activities such as antioxidant, anticancer, and antimalarial activities. The transcriptional regulation of flavonoid biosynthesis in A. annua has not been well-studied. In this study, we identified a YABBY family transcription factor, AaYABBY5, as a positive regulator of anthocyanin and total flavonoid contents in A. annua. AaYABBY5 was selected based on its similar expression pattern to the phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and flavonol synthase (FLS) genes. A transient dual-luciferase assay in Nicotiana bethamiana with the AaYABBY5 effector showed a significant increase in the activity of the downstream LUC gene, with reporters AaPAL, AaCHS, AaCHI, and AaUFGT. The yeast one-hybrid system further confirmed the direct activation of these promoters by AaYABBY5. Gene expression analysis of stably transformed AaYABBY5 overexpression, AaYABBY5 antisense, and control plants revealed a significant increase in the expression of AaPAL, AaCHS, AaCHI, AaFLS, AaFSII, AaLDOX, and AaUFGT in AaYABBY5 overexpression plants. Moreover, their total flavonoid content and anthocyanin content were also found to increase. AaYABBY5 antisense plants showed a significant decrease in the expression of flavonoid biosynthetic genes, as well as a decrease in anthocyanin and total flavonoid contents. In addition, phenotypic analysis revealed deep purple-pigmented stems, an increase in the leaf lamina size, and higher trichome densities in AaYABBY5 overexpression plants. Together, these data proved that AaYABBY5 is a positive regulator of flavonoid biosynthesis in A. annua. Our study provides candidate transcription factors for the improvement of flavonoid concentrations in A. annua and can be further extended to elucidate its mechanism of regulating trichome development.

A Novel Spo11 Homologue Functions as a Positive Regulator in Cyst Differentiation in Giardia lamblia

Giardia lamblia persists in a dormant state with a protective cyst wall for transmission. It is incompletely known how three cyst wall proteins (CWPs) are coordinately synthesized during encystation. Meiotic recombination is required for sexual reproduction in animals, fungi, and plants. It is initiated by formation of double-stranded breaks by a topoisomerase-like Spo11. It has been shown that exchange of genetic material in the fused nuclei occurs during Giardia encystation, suggesting parasexual recombination processes of this protozoan. Giardia possesses an evolutionarily conserved Spo11 with typical domains for cleavage reaction and an upregulated expression pattern during encystation. In this study, we asked whether Spo11 can activate encystation process, like other topoisomerases we previously characterized. We found that Spo11 was capable of binding to both single-stranded and double-stranded DNA in vitro and that it could also bind to the cwp promoters in vivo as accessed in chromatin immunoprecipitation assays. Spo11 interacted with WRKY and MYB2 (named from myeloblastosis), transcription factors that can activate cwp gene expression during encystation. Interestingly, overexpression of Spo11 resulted in increased expression of cwp1-3 and myb2 genes and cyst formation. Mutation of the Tyr residue for the active site or two conserved residues corresponding to key DNA-binding residues for Arabidopsis Spo11 reduced the levels of cwp1-3 and myb2 gene expression and cyst formation. Targeted disruption of spo11 gene with CRISPR/Cas9 system led to a significant decrease in cwp1-3 and myb2 gene expression and cyst number. Our results suggest that Spo11 acts as a positive regulator for Giardia differentiation into cyst.

Transcription Factor TaWRKY51 Is a Positive Regulator in Root Architecture and Grain Yield Contributing Traits

Wheat is one of the staple food crops. The utilization of elite genetic resources to develop resource-efficient wheat varieties is an effective approach to deal with the challenges of climate change and population growth. WRKY transcription factors (TFs) are multifaceted regulators of plant growth and development and response to environmental stress. The previous studies have shown that TaWRKY51 positively regulates the development of lateral roots, while its roles in agronomic trait development are not clear, and there is no functional marker for molecular breeding. To bridge the gap, we cloned the three members of TaWRKY51 and found they were highly expressed in the roots and flag leaves at the flowering stage and were induced by the multiple abiotic stresses and phytohormones. The highest expression level was observed in TaWRKY51-2D, followed by TaWRKY51-2A and -2B. The two haplotypes/alleles for each member were identified in the natural populations, and functional markers were developed accordingly. The association assays revealed that Hap-2A-I was an elite haplotype for the large spike, Hap-2B-II and allele-G were favorable haplotypes/alleles for long root. However, only Hap-2A-I was selected for wheat breeding in China. The results of transgenic experiments showed that the rice lines overexpressing TaWRKY51 had large panicle, high thousand-grain-weight, and more crown and lateral roots, which further confirmed the results of association analysis. In short, TaWRKY51 is a positive regulator of the root architecture and grain yield (GY) contributing traits. The elite gene resources and functional markers may be utilized in the marker-assisted selection for high-yield breeding in wheat.

GhWRKY1-like, a WRKY transcription factor, mediates drought tolerance in Arabidopsis via modulating ABA biosynthesis

Background Drought stress has great negative effects on the plant growth and development. The tolerance of plants to such abiotic stress is triggered by complicated and multilayered signaling pathways to restore cellular homeostasis and to promote survival. The WRKY family is one of the largest transcription factor families in higher plants, and has been well recognized for the roles in regulating plants tolerance to abiotic and biotic stress. However, little is known about how the WRKY genes regulate drought resistance in cotton. Results In this work, we identified the WRKY transcription factor GhWRKY1-like from upland cotton as a positive regulator of tolerance to drought that directly manipulates abscisic acid (ABA) biosynthesis. Overexpression of GhWRKY1-like in Arabidopsis constitutively activated ABA biosynthesis genes, signaling genes, responsive genes and drought related maker genes, and led to enhanced tolerance to drought. Further analysis has shown that GhWRKY1-like can interact with “W-box” cis-elements of the promoters of AtNCED2, AtNCED5, AtNCED6 and AtNCED9 which are essential enzymes for ABA biosynthesis, and promotes the expression of those target genes. Conclusions In summary, our findings suggest that GhWRKY1-like may act as a positive regulator in Arabidopsis tolerance to drought via directly interacting with the promoters of AtNCED2, AtNCED5, AtNCED6 and AtNCED9 to promote ABA biosynthesis.