Dehydrin Client Proteins Identified Using Phage Display Affinity Selected Libraries Processed With Paired-End Phage Sequencing.

The late embryogenesis abundant proteins (LEAPs) are a class of noncatalytic, intrinsically disordered proteins with a malleable structure. Some LEAPs exhibit a protein and/or membrane binding capacity and LEAP binding to various targets has been positively correlated with abiotic stress tolerance. Regarding the LEAPs' presumptive role in protein protection, identifying client proteins (CtPs) to which LEAPs bind is one practicable means of revealing the mechanism by which they exert their function.

Natural variation of CT2 affects the embryo/kernel weight ratio in maize.

Embryo size is a critical trait determining not only grain yield but also the nutrition of the maize kernel. Up to the present, only a few genes have been characterized affecting the maize embryo/kernel ratio. Here, we identify 63 genes significantly associated with maize embryo/kernel weight ratio using a genome-wide association study (GWAS).

Raffinose catabolism enhances maize waterlogging tolerance by stimulating adventitious root growth and development.

Raffinose mitigates plant heat, drought, and cold stresses; however, whether raffinose contributes to plant waterlogging tolerance is unknown. The maize raffinose synthase mutant zmrafs-1 had seedlings that lack raffinose, generated fewer and shorter adventitious roots, and were more sensitive to waterlogging stress, while overexpression of the raffinose synthase gene, ZmRAFS, increased raffinose content, stimulated adventitious root formation, and enhanced waterlogging tolerance of maize seedlings. Transcriptome analysis of null segregant seedlings compared with zmrafs-1, particularly when waterlogged, revealed that the expression of genes related to galactose metabolism and the auxin biosynthetic pathway were up-regulated by raffinose.

Maize PIMT2 repairs damaged 3-METHYLCROTONYL COA CARBOXYLASE in mitochondria, affecting seed vigor.

PROTEIN l-ISOASPARTYL O-METHYLTRANSFERASE (PIMT) affects seed vigor by repairing damaged proteins. While PIMT is capable of isoaspartyl (isoAsp) repair in all proteins, those proteins most susceptible to isoAsp formation have not been well characterized, and the mechanisms by which PIMT affects seed vigor remain largely unknown. Using co-immunoprecipitation and LC-MS/MS, we found that maize (Zea mays) PIMT2 (ZmPIMT2) interacted predominantly with both subunits of maize 3-METHYLCROTONYL COA CARBOXYLASE (ZmMCC).

Raffinose positively regulates maize drought tolerance by reducing leaf transpiration.

Drought stress is one of the major constraints of global crop production. Raffinose, a non-reducing trisaccharide, has been considered to regulate positively the plant drought stress tolerance; however, evidence that augmenting raffinose production in leaves results in enhanced plant drought stress tolerance is lacking. The biochemical mechanism through which raffinose might act to mitigate plant drought stress remains unidentified.

ZmAGA1 Hydrolyzes RFOs Late during the Lag Phase of Seed Germination, Shifting Sugar Metabolism toward Seed Germination Over Seed Aging Tolerance.

Raffinose family oligosaccharides (RFOs) are accumulated during the late stage of seed development and hydrolyzed during seed germination. The process of raffinose hydrolysis during seed germination and how this process affects seed vigor remains unknown. We report here that maize alkaline α-galactosidase 1 (ZmAGA1) protein is translationally induced and is capable of hydrolyzing RFOs as well as a precursor, galactinol, during seed germination.

ZmDREB2A regulates ZmGH3.2 and ZmRAFS, shifting metabolism towards seed aging tolerance over seedling growth.

Seed aging tolerance and rapid seedling growth are important agronomic traits for crop production; however, how these traits are controlled at the molecular level remains largely unknown. The unaged seeds of two independent maize DEHYDRATION-RESPONSIVE ELEMENT-BINDING2A mutant (zmdreb2a) lines, with decreased expression of GRETCHEN HAGEN3.2 (ZmGH3.

Direct and indirect targets of the arabidopsis seed transcription factor ABSCISIC ACID INSENSITIVE3.

Arabidopsis thaliana ABSCISIC ACID INSENSITIVE3 (ABI3) is a transcription factor in the B3 domain family. ABI3, along with B3 domain transcription factors LEAFY COTYLEDON2 (LEC2) and FUSCA3 (FUS3), and LEC1, a subunit of the CCAAT box-binding complex, form the so-called LAFL network to control various aspects of seed development and maturation. ABI3 also contributes to the abscisic acid (ABA) response.

Raffinose synthase enhances drought tolerance through raffinose synthesis or galactinol hydrolysis in maize and plants.

Raffinose and its precursor galactinol accumulate in plant leaves during abiotic stress. RAFFINOSE SYNTHASE (RAFS) catalyzes raffinose formation by transferring a galactosyl group of galactinol to sucrose. However, whether RAFS contributes to plant drought tolerance and, if so, by what mechanism remains unclear.

ZmDREB1A Regulates RAFFINOSE SYNTHASE Controlling Raffinose Accumulation and Plant Chilling Stress Tolerance in Maize.

Raffinose accumulation is positively correlated with plant chilling stress tolerance; however, the understanding of the function and regulation of raffinose metabolism under chilling stress remains in its infancy. RAFFINOSE SYNTHASE (RAFS) is the key enzyme for raffinose biosynthesis. In this study, we report that two independent maize (Zea mays) zmrafs mutant lines, in which raffinose was completely abolished, were more sensitive to chilling stress and their net photosynthetic product (total soluble sugars and starch) accumulation was significantly decreased compared with controls after chilling stress.

Maize HSFA2 and HSBP2 antagonistically modulate raffinose biosynthesis and heat tolerance in Arabidopsis.

Raffinose is thought to play an important role in plant tolerance of abiotic stress. We report here that maize HEAT SHOCK FACTOR A2 (ZmHSFA2) and HEAT SHOCK BINDING PROTEIN 2 (ZmHSBP2) physically interact with each other and antagonistically modulate expression of GALACTINOL SYNTHASE2 (ZmGOLS2) and raffinose biosynthesis in transformed maize protoplasts and Arabidopsis plants. Overexpression of ZmHSFA2 in Arabidopsis increased the expression of Arabidopsis AtGOLS1, AtGOLS2 and AtRS5 (RAFFINOSE SYNTHASE), increased the raffinose content in leaves and enhanced plant heat stress tolerance.

Maize VIVIPAROUS1 Interacts with ABA INSENSITIVE5 to Regulate GALACTINOL SYNTHASE2 Expression Controlling Seed Raffinose Accumulation.

Raffinose, an oligosaccharide found in many seeds, plays an important role in seed vigor; however, the regulatory mechanism governing raffinose biosynthesis remains unclear. We report here that maize W22 wild type (WT) seeds, but not W22 viviparous1 ( zmvp1) mutant seeds, start accumulating galactinol and raffinose 28 days after pollination (DAP). Transcriptome analysis of the zmvp1 embryo showed that the expression of GALACTINOL SYNTHASE2 ( GOLS2) was down-regulated relative to WT.

PHYTOCHROME INTERACTING FACTOR1 interactions leading to the completion or prolongation of seed germination.

In Arabidopsis thaliana, the basic Helix Loop Helix transcription factor, PHYTOCHROME INTERACTING FACTOR1 (PIF1) is known to orchestrate the seed transcriptome such that, ultimately, proteins repressing the completion of germination are produced in darkness. While PIF1-mediated control of abscisic acid (ABA) and gibberellic acid (GA) anabolism/catabolism is indirect, PIF1 action favors ABA while discriminating against GA, firmly establishing ABA's repressive influence on the completion of germination. The result is tissue that is more sensitive to and producing more ABA; and is less responsive to and deficient in GA.

KELCH F-BOX protein positively influences Arabidopsis seed germination by targeting PHYTOCHROME-INTERACTING FACTOR1.

Seeds employ sensory systems that assess various environmental cues over time to maximize the successful transition from embryo to seedling. Here we show that the F-BOX protein COLD TEMPERATURE-GERMINATING (CTG)-10, identified by activation tagging, is a positive regulator of this process. When overexpressed (OE), CTG10 hastens aspects of seed germination.

Regulation of Seed Vigor by Manipulation of Raffinose Family Oligosaccharides in Maize and Arabidopsis thaliana.

Raffinose family oligosaccharides (RFOs) accumulate in seeds during maturation desiccation in many plant species. However, it remains unclear whether RFOs have a role in establishing seed vigor. GALACTINOL SYNTHASE (GOLS), RAFFINOSE SYNTHASE (RS), and STACHYOSE SYNTHASE (STS) are the enzymes responsible for RFO biosynthesis in plants.

ZmGOLS2, a target of transcription factor ZmDREB2A, offers similar protection against abiotic stress as ZmDREB2A.

GALACTINOL SYNTHASE is the first committed enzyme in the raffinose biosynthetic pathway. We have previously characterized the maize (Zea mays) GALACTINOL SYNTHASE2 gene (ZmGOLS2) as abiotic stress induced. To further investigate the regulation of ZmGOLS2 gene expression, individual luciferase expression vectors,in which the luciferase gene was controlled by different lengths of the ZmGOLS2 promoter, were co-transfected into maize protoplasts with either a ZmDREB2A- or a GFP-expression vector.

A role for barley CRYPTOCHROME1 in light regulation of grain dormancy and germination.

It is well known that abscisic acid (ABA) plays a central role in the regulation of seed dormancy and that transcriptional regulation of genes encoding ABA biosynthetic and degradation enzymes is responsible for determining ABA content. However, little is known about the upstream signaling pathways impinging on transcription to ultimately regulate ABA content or how environmental signals (e.g.

A protocol for phage display and affinity selection using recombinant protein baits.

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein.

Functional analysis of the 5' regulatory region of the maize GALACTINOL SYNTHASE2 gene.

Maize (Zea mays) GALACTINOL SYNTHASE (GolS) is a key enzyme in the raffinose biosynthetic pathway. We have previously characterized the maize GolS2 (ZmGolS2) gene as heat shock induced in maize germinating seeds and cultured cells. Here we report the identification, isolation and characterization of the 1.

Sorghum mutant RG displays antithetic leaf shoot lignin accumulation resulting in improved stem saccharification properties.

Background: Improving saccharification efficiency in bioenergy crop species remains an important challenge. Here, we report the characterization of a Sorghum (Sorghum bicolor L.) mutant, named REDforGREEN (RG), as a bioenergy feedstock.

Uses of phage display in agriculture: sequence analysis and comparative modeling of late embryogenesis abundant client proteins suggest protein-nucleic acid binding functionality.

A group of intrinsically disordered, hydrophilic proteins-Late Embryogenesis Abundant (LEA) proteins-has been linked to survival in plants and animals in periods of stress, putatively through safeguarding enzymatic function and prevention of aggregation in times of dehydration/heat. Yet despite decades of effort, the molecular-level mechanisms defining this protective function remain unknown. A recent effort to understand LEA functionality began with the unique application of phage display, wherein phage display and biopanning over recombinant Seed Maturation Protein homologs from Arabidopsis thaliana and Glycine max were used to retrieve client proteins at two different temperatures, with one intended to represent heat stress.

An Arabidopsis ATP-dependent, DEAD-box RNA helicase loses activity upon IsoAsp formation but is restored by PROTEIN ISOASPARTYL METHYLTRANSFERASE.

Orthodox seeds are capable of withstanding severe dehydration. However, in the dehydrated state, Asn and Asp residues in proteins can convert to succinimide residues that can further react to predominantly form isomerized isoAsp residues upon rehydration (imbibition). IsoAsp residues can impair protein function and can render seeds nonviable, but PROTEIN ISOASPARTYL METHYLTRANSFERASE (PIMT) can initiate isoAsp conversion to Asp residues.

Uses of phage display in agriculture: a review of food-related protein-protein interactions discovered by biopanning over diverse baits.

This review highlights discoveries made using phage display that impact the use of agricultural products. The contribution phage display made to our fundamental understanding of how various protective molecules serve to safeguard plants and seeds from herbivores and microbes is discussed. The utility of phage display for directed evolution of enzymes with enhanced capacities to degrade the complex polymers of the cell wall into molecules useful for biofuel production is surveyed.

The role of SORBITOL DEHYDROGENASE in Arabidopsis thaliana.

SORBITOL DEHYDROGENASE (SDH, EC 1.1.1.

Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment.

Arabidopsis (Arabidopsis thaliana) epidermal seed coat cells follow a complex developmental program where, following fertilization, cells of the ovule outer integument differentiate into a unique cell type. Two hallmarks of these cells are the production of a doughnut-shaped apoplastic pocket filled with pectinaceous mucilage and the columella, a thick secondary cell wall. Cellulose is thought to be a key component of both these secondary cell wall processes.