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Abstract Background The maize (Zea mays) red aleurone1 (pr1) encodes a CYP450-dependent flavonoid 3’-hydroxylase (ZmF3’H1) required for the biosynthesis of purple and red anthocyanin pigments. We previously showed that Zmf3’h1 is regulated by C1 (Colorless1) and R1 (Red1) transcription factors. The current study demonstrates that, in addition to its role in anthocyanin biosynthesis, the Zmf3’h1 gene also participates in the biosynthesis of 3-deoxyflavonoids and phlobaphenes that accumulate in maize pericarps, cob glumes, and silks. Biosynthesis of 3-deoxyflavonoids is regulated by P1 (Pericarp color1) and is independent from the action of C1 and R1 transcription factors. Results In maize, apiforol and luteoforol are the precursors of condensed phlobaphenes. Maize lines with functional alleles of pr1 and p1 (Pr1;P1) accumulate luteoforol, while null pr1 lines with a functional or non-functional p1 allele (pr1;P1 or pr1;p1) accumulate apiforol. Apiforol lacks a hydroxyl group at the 3’-position of the flavylium B-ring, while luteoforol has this hydroxyl group. Our biochemical analysis of accumulated compounds in different pr1 genotypes showed that the pr1 encoded ZmF3’H1 has a role in the conversion of mono-hydroxylated to bi-hydroxylated compounds in the B-ring. Steady state RNA analyses demonstrated that Zmf3’h1 mRNA accumulation requires a functional p1 allele. Using a combination of EMSA and ChIP experiments, we established that the Zmf3’h1 gene is a direct target of P1. Highlighting the significance of the Zmf3’h1 gene for resistance against biotic stress, we also show here that the p1 controlled 3-deoxyanthocyanidin and C-glycosyl flavone (maysin) defence compounds accumulate at significantly higher levels in Pr1 silks as compared to pr1 silks. By virtue of increased maysin synthesis in Pr1 plants, corn ear worm larvae fed on Pr1; P1 silks showed slower growth as compared to pr1; P1 silks. Conclusions Our results show that the Zmf3’h1 gene participates in the biosynthesis of phlobaphenes and agronomically important 3-deoxyflavonoid compounds under the regulatory control of P1.

Cold stress resulting from chilling and freezing temperatures substantially reduces crop production worldwide. To identify genes critical for cold tolerance in plants, we screened Arabidopsis thaliana mutants for deregulated expression of a firefly luciferase reporter gene under the control of the C-REPEAT BINDING FACTOR2 (CBF2) promoter (CBF2 : LUC). A regulator of CBF gene expression1 (rcf1-1) mutant that is hypersensitive to cold stress was chosen for in-depth characterization. RCF1 encodes a cold-inducible DEAD (Asp-Glu-Ala-Asp) box RNA helicase. Unlike a previously reported DEAD box RNA helicase (LOW EXPRESSION OF OSMOTICALLY RESPONSIVE GENES4 [LOS4]) that regulates mRNA export, RCF1 does not play a role in mRNA export. Instead, RCF1 functions to maintain proper splicing of pre-mRNAs; many cold-responsive genes are mis-spliced in rcf1-1 mutant plants under cold stress. Functional characterization of four genes (PSEUDO-RESPONSE REGULATOR5 [ PRR5 ], SHAGGY-LIKE SERINE/THREONINE KINASE12 [ SK12 ], MYB FAMILY TRANSCRIPTION FACTOR CIRCADIAN1 [ CIR1 ], and SPFH/PHB DOMAIN-CONTAINING MEMBRANE-ASSOCIATED PROTEIN [ SPFH ]) that are mis-spliced in rcf1-1 revealed that these genes are cold-inducible positive (CIR1 and SPFH) and negative (PRR5 and SK12) regulators of cold-responsive genes and cold tolerance. Together, our results suggest that the cold-inducible RNA helicase RCF1 is essential for pre-mRNA splicing and is important for cold-responsive gene regulation and cold tolerance in plants. [ABSTRACT FROM AUTHOR]

For the past two decades, genetically encoded fluorescent proteins have emerged as the most popular method to image the plant cytoskeleton. Because fluorescent protein technology involves handling living plant cells, it is important to implement protocols that enable these delicate plant specimens to maintain optimal growth for the entire duration of the imaging experiment. To this end, we rely on a system that consists of a large coverslip coated with nutrient-supplemented agar. This agar-coverslip system is planted with surface-sterilized Arabidopsis thaliana seeds expressing cytoskeletal fluorescent protein reporters. The agar-coverslip system with planted seeds is then maintained in an environmentally controlled growth chamber. The entire setup is transferred onto the stage of a confocal microscope for imaging when roots of germinated seedlings reach a desired length. For plants with larger roots such as Medicago truncatula, the polymerized nutrient-supplemented agar is gently lifted or cut and used to secure pre-germinated seeds on the coverslip prior to root imaging. The agar-coverslip system we use for imaging the cytoskeleton in living roots along with general methods for expressing green fluorescent protein (GFP)-based cytoskeletal reporters in hairy roots of Medicago truncatula is described here., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

DNA-binding proteins, including transcription factors, play essential roles in many biological processes. The identification of the DNA sequences to which these proteins bind is a first, yet still challenging, step for determining their functions. SELEX provides an excellent tool for deciphering protein DNA-binding sequence specificity, and it has been widely adopted for addressing fundamental biological questions. SELEX is an experimental procedure that involves the progressive selection, from a large combinatorial double-stranded oligonucleotide library, of DNA ligands with variable DNA-binding affinities and specificities by repeated rounds of partition and amplification. In this chapter, we describe a SELEX protocol that we have successfully applied to both plant and animal MYB transcription factors.