Identification of Novel Growth Regulators in Plant Populations Expressing Random Peptides.

The use of chemical genomics approaches allows the identification of small molecules that integrate into biological systems, thereby changing discrete processes that influence growth, development, or metabolism. Libraries of chemicals are applied to living systems, and changes in phenotype are observed, potentially leading to the identification of new growth regulators. This work describes an approach that is the nexus of chemical genomics and synthetic biology. Here, each plant in an extensive population synthesizes a unique small peptide arising from a transgene composed of a randomized nucleic acid sequence core flanked by translational start, stop, and cysteine-encoding (for disulfide cyclization) sequences. Ten and 16 amino acid sequences, bearing a core of six and 12 random amino acids, have been synthesized in Arabidopsis (Arabidopsis thaliana) plants. Populations were screened for phenotypes from the seedling stage through senescence. Dozens of phenotypes were observed in over 2,000 plants analyzed. Ten conspicuous phenotypes were verified through separate transformation and analysis of multiple independent lines. The results indicate that these populations contain sequences that often influence discrete aspects of plant biology. Novel peptides that affect photosynthesis, flowering, and red light response are described. The challenge now is to identify the mechanistic integrations of these peptides into biochemical processes. These populations serve as a new tool to identify small molecules that modulate discrete plant functions that could be produced later in transgenic plants or potentially applied exogenously to impart their effects. These findings could usher in a new generation of agricultural growth regulators, herbicides, or defense compounds.

Validation of reference transcripts in strawberry (Fragaria spp.).

Contemporary methods to assay gene expression depend on a stable set of reference transcripts for accurate quantitation. A lack of well-tested reference genes slows progress in characterizing gene expression in high-value specialty crops. In this study, a set of strawberry (Fragaria spp.) constitutively expressed reference genes has been identified by merging digital gene expression data with expression profiling. Constitutive reference candidates were validated using quantitative PCR and hybridization. Several transcripts have been identified that show improved stability across tissues relative to traditional reference transcripts. Results are similar between commercial octoploid strawberry and the diploid model. Our findings also show that while some never-before-used references are appropriate for most applications, even the most stable reference transcripts require careful assessment across the diverse tissues and fruit developmental states before being adopted as controls.

A synthetic peptide encoded by a random DNA sequence inhibits discrete red light responses.

We have identified a synthetic peptide that interrupts discrete aspects of seedling development under red light. Previous reports have demonstrated that plants transformed with random DNA sequences produce synthetic peptides that affect plant biology. In this report, one specific peptide is characterized that inhibits discrete aspects of red light-mediated photomorphogenic development in Arabidopsis thaliana . Seedlings expressing the PEP6-32 peptide presented longer hypocotyls and diminished cotyledon expansion when grown under red light. Other red light-mediated seedling processes such as induction of Lhcb (cab) transcripts or loss of vertical growth remained unaffected. Long-term responses to red light in PEP6-32 expressing plants, such as repression of flowering time, did not show defects in red light signaling or integration. A synthesized peptide applied exogenously induced the long-hypocotyl phenotype under red light in non-transformed seedlings. The results indicate that the PEP6-32 peptide causes discrete cell expansion abnormalities during early seedling development in red light that mimic weak phyB alleles, yet only in some aspects of seedling photomorphogenesis. The findings demonstrate that new chemistries derived from random peptide expression can modulate specific facets of plant growth and development.

A brittle-2 transgene increases maize yield by acting in maternal tissues to increase seed number.

The enzyme ADP-glucose pyrophosphorylase is essential for starch biosynthesis and is highly regulated. Here, mutations that increased heat stability and interactions with allosteric effectors were incorporated into the small subunit of the isoform known to be expressed at high levels in the maize endosperm. The resulting variants were transformed into maize with expression targeted to the endosperm. Transgenes harboring the changes increased yield some 35%; however, yield enhancement occurred via an increase in seed number rather than by increased seed weight. Interestingly, seed number increase is controlled by the genotype of the plant rather than the genotype of the seed as seeds increase in number whether or not they contain the transgene as long as the maternal parent has the transgene. The transgene is however expressed in the endosperm, and the altered allosteric and stability properties initially seen in Escherichia coli expression experiments are also seen with the endosperm-expressed gene. The extent of seed number increase is positively correlated with the average daily high temperature during the first 4 days postpollination. While these results were unexpected, they echo the phenotypic changes caused by the insertion of an altered large subunit of this enzyme reported previously (Plant Cell, 24, 2012, 2352). These results call into question some of the reported fundamental differences separating starch synthesis in the endosperm vis-à-vis other plant tissues.

Analysis of Block of cell proliferation 1 (BOP1) activity in strawberry and Arabidopsis.

Block of cell proliferation (BOP) proteins are conserved among eukaryotes, and studies in mammals and yeast have described their role in ribosome biogenesis and cell cycle regulation. A BOP1 orthologue was identified in plants, and loss-of-function analyses in tobacco cells confirmed similar activities. This report characterizes a role for BOP1 activity in planta. Two transgenic plant species were used: the diploid strawberry (Fragaria vesca) and Arabidopsis thaliana. FvBOP1 silencing showed changes in pre-rRNA processing, and demonstrated FvBOP1's role in growth and physiology throughout different stages of plant development. In the strawberry, repression of FvBOP1 activity decreased plant fitness prior to flowering, followed by plant death after the reproductive transition, indicating that BOP1 activity is required for transition back to vegetative growth after flowering. A T-DNA null allele of the AtBOP1 gene is lethal, and a 50% decrease in transcript accumulation is sufficient to cause severe developmental defects linked to defective cell division. The conserved protein BOP1 is essential for viability. Lower transcript levels result in defects in rRNA processing and developmental abnormalities that are consistent with its predicted role in ribosome biogenesis.

A strawberry KNOX gene regulates leaf, flower and meristem architecture.

The KNOTTED-LIKE HOMEODOMAIN (KNOX) genes play a central role in maintenance of the shoot apical meristem. They also contribute to the morphology of simple and compound leaves. In this report we characterize the FaKNOX1 gene from strawberry (Fragaria spp.) and demonstrate its function in trasgenic plants. The FaKNOX1 cDNA was isolated from a cultivated strawberry (F.×ananassa) flower EST library. The sequence is most similar to Class I KNOX genes, and was mapped to linkage group VI of the diploid strawberry genome. Unlike most KNOX genes studied, steady-state transcript levels were highest in flowers and fruits. Transcripts were also detected in emerging leaf primordia and the apical dome. Transgenic strawberry plants suppressing or overexpressing FaKNOX1 exhibited conspicuous changes in plant form. The FaKNOX1 RNAi plants presented a dwarfed phenotype with deeply serrated leaflets and exaggerated petiolules. They also exhibited a high level of cellular disorganization of the shoot apical meristem and leaves. Overexpression of FaKNOX1 caused dwarfed stature with wrinkled leaves. These gain- and loss-of-function assays in strawberry functionally demonstrate the contributions of a KNOX domain protein in a rosaceous species.