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Mannans are hemicellulosic polysaccharides that have a structural role and serve as storage reserves during plant growth and development. Previous studies led to the conclusion that mannan synthase enzymes in several plant species are encoded by members of the cellulose synthase-like A (CSLA) gene family. Arabidopsis has nine members of the CSLA gene family. Earlier work has shown that CSLA9 is responsible for the majority of glucomannan synthesis in both primary and secondary cell walls of Arabidopsis inflorescence stems. Little is known about how expression of the CLSA9 gene is regulated. Sequence analysis of the CSLA9 promoter region revealed the presence of multiple copies of a cis-regulatory motif (M46RE) recognized by transcription factor MYB46, leading to the hypothesis that MYB46 (At5g12870) is a direct regulator of the mannan synthase CLSA9. We obtained several lines of experimental evidence in support of this hypothesis. First, the expression of CSLA9 was substantially upregulated by MYB46 overexpression. Second, electrophoretic mobility shift assay (EMSA) was used to demonstrate the direct binding of MYB46 to the promoter of CSLA9 in vitro. This interaction was further confirmed in vivo by a chromatin immunoprecipitation assay. Finally, over-expression of MYB46 resulted in a significant increase in mannan content. Considering the multifaceted nature of MYB46-mediated transcriptional regulation of secondary wall biosynthesis, we reasoned that additional transcription factors are involved in the CSLA9 regulation. This hypothesis was tested by carrying out yeast-one hybrid screening, which identified ANAC041 and bZIP1 as direct regulators of CSLA9. Transcriptional activation assays and EMSA were used to confirm the yeast-one hybrid results. Taken together, we report that transcription factors ANAC041, bZIP1 and MYB46 directly regulate the expression of CSLA9.

A pectin methylesterase inhibitor (SolyPMEI) from tomato has been identified and characterised by a functional genomics approach. SolyPMEI is a cell wall protein sharing high similarity with Actinidia deliciosa PMEI (AdPMEI), the best characterised inhibitor from kiwi. It typically affects the activity of plant pectin methylesterases (PMEs) and is inactive against a microbial PME. SolyPMEI transcripts were mainly expressed in flower, pollen and ripe fruit where the protein accumulated at breaker and turning stages of ripening. The expression of SolyPMEI correlated during ripening with that of PME-1, the major fruit specific PME isoform. The interaction of SolyPMEI with PME-1 was demonstrated in ripe fruit by gel filtration and by immunoaffinity chromatography. The analysis of the zonal distribution of PME activity and the co-localization of SolyPMEI with high esterified pectins suggest that SolyPMEI regulates the spatial patterning of distribution of esterified pectins in fruit.

Plant science has made considerable progress in developing new biotechnology-based plant breeding techniques to alter genetic and epigenetic factors. In this chapter, we will discuss novel plant products (NPPs) obtained by cisgenesis, intragenesis and genome engineering using site-specific nucleases and gene-targeting oligonucleotides. Among these, zinc finger nucleases, transcription activator-like effector nucleases and clustered, regularly interspaced, short palindromic repeats-associated Cas9 nucleases. Reverse breeding methods and backcrossing of the engineered plants with natural varieties has provided improved plants and fruit trees devoid of transgenes and cisgenes. There is an increasing higher public acceptance of the NPPs devoid of virus sequences and antibiotic genes, and containing only genetic material derived from the species itself or from closely related species. An overview is presented on the differences between regulation and regulatory bodies in various countries, with a need for harmonization in the ruling and in definition differences between modified and non-modified plant genomes.

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica–specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus., Author Summary Algae are a highly diverse group of organisms that have become the focus of renewed interest due to their potential for producing biofuel feedstocks, nutraceuticals, and biomaterials. Their high photosynthetic yields and ability to grow in areas unsuitable for agriculture provide a potential sustainable alternative to using traditional agricultural crops for biofuels. Because none of the algae currently in use have a history of domestication, and bioengineering of algae is still in its infancy, there is a need to develop algal strains adapted to cultivation for industrial large-scale production of desired compounds. Model organisms ranging from mice to baker's yeast have been instrumental in providing insights into fundamental biological structures and functions. The algal field needs versatile models to develop a fundamental understanding of photosynthetic production of biomass and valuable compounds in unicellular, marine, oleaginous algal species. To contribute to the development of such an algal model system for basic discovery, we sequenced the genome and two sets of transcriptomes of N. oceanica CCMP1779, assembled the genomic sequence, identified putative genes, and began to interpret the function of selected genes. This species was chosen because it is readily transformable with foreign DNA and grows well in culture.