**4. Conclusions and future perspectives**

Due to the increasing concerns on the environment, climate change, and limited natural resources, there is considerable effort applied to produce chemicals and materials from renewable biomass. While initial emphasis was reversed on biofuel production from food plant sugars, the competition between crop usage for food and non-food applications has stimulated research efforts to genetically improve yield and quality-related traits of plants for biorefining applications.

Targeted genetic improvement of biomass for biorefining applications depends on identifying genetic variation in critical morphological, structural, biochemical, and physiological traits affecting biomass yield and its chemical composition. This involves manipulating complex traits, such as those associated with plant growth and development or tolerances to abiotic and biotic stresses, usually in production environments that are highly variable and unpre‐ dictable, as well as to gain insights into the less digestible carbohydrates in cell walls compo‐ nents. This last aspect, in turn, has promoted inspections on the use of other plant-derived metabolites for chemical productions embracing the high-value specialty segments via platform intermediates needed for bulk production.

Although there is genetic variability in the traits sustaining biomass yield, many of them important in crop productivity and sustainability are complex multigene traits, often difficult to breed for. Nevertheless, advances in plant genetics and genomic technologies are contribu‐ ting to the acceleration of gene discovery for product development. In this respect, several new genomics technologies such as next-generation sequencing and high-throughput marker genotyping can be used not only for developing high-density genetic and physical maps but also for generating transcriptome or sequence data [55]. These approaches together with – omics technologies (e.g., transcriptomics, genetical genomics, metabolomics, and proteomics) have emerged as powerful tools for understanding genome variation in crop species at DNA, RNA, as well as protein level and for identifying genomic regions or genes affecting the expression of trait(s) that are of interest to improving plants for biorefining applications or for breeding varieties with in-built new traits such as creating higher value-added products. Furthermore, a range of novel genetic techniques, particularly techniques collectively referred to as 'genome editing', have been developed that allow targeted changes to be made to genomes [103]. Changes can include adding or removing DNA at a specified location in the genome or replacing a specified segment of DNA with a different one. It is also expected to make epigenetic changes (histone modifications and DNA methylation), where the DNA sequence remains unchanged but gene expression is altered because of chromatin changes that may be heritable [104].

miR319a reduce organ size and cause premature cell differentiation [93, 94]. In fact, promoting cell differentiation has been proposed as the primary function of TCPs, rather than directly

A novel gene regulation mechanism was recently discovered in metazoans, the RNA silencing or RNAi, or microRNA (miRNAs) [96]. Rodriguez et al. [97] found that overexpression of miR396 in Arabidopsis had a negative impact on cell proliferation in developing leaves through the repression of GRF activity and a decrease in the expression of cell cycle genes. Accordingly, disruption of the recognition of *GRF2* by miR396 resulted in larger leaves with more cells than control plants. Similarly in Arabidopsis, it was reported that over-expression of miR156 promotes a vegetative-phase transition delay and an enhancement in leaf initiation rate [98], while over-expression of miR172 induces adult leaf traits and flowering [99]. The regulation of both miRNAs is closely connected as their expression is influenced by age, temperature, and light acting in a contrasting way [100]. At a molecular level, miR156 acts through the down regulation of genes coding for the *SQUAMOSA PROMOTER BINDING-LIKE (SPL*) and the *AP2-like transcription factor*, respectively, are subjected to feedback regula‐

Recently, miRNAs have also emerged as key regulators of phytohormone response pathways *in planta* by affecting their metabolism, distribution, and perception [100,102] have demon‐ strated that gibberellic acid (GA) promotes flowering in Arabidopsis through a miR156 dependent pathway, indicating a connection between miRNA and phytohormone signaling pathways in the control of shoot development. In general, it appears evident from this data that miRNAs as growth regulators are interesting targets for gene manipulation for improving

Due to the increasing concerns on the environment, climate change, and limited natural resources, there is considerable effort applied to produce chemicals and materials from renewable biomass. While initial emphasis was reversed on biofuel production from food plant sugars, the competition between crop usage for food and non-food applications has stimulated research efforts to genetically improve yield and quality-related traits of plants for biorefining

Targeted genetic improvement of biomass for biorefining applications depends on identifying genetic variation in critical morphological, structural, biochemical, and physiological traits affecting biomass yield and its chemical composition. This involves manipulating complex traits, such as those associated with plant growth and development or tolerances to abiotic and biotic stresses, usually in production environments that are highly variable and unpre‐ dictable, as well as to gain insights into the less digestible carbohydrates in cell walls compo‐ nents. This last aspect, in turn, has promoted inspections on the use of other plant-derived metabolites for chemical productions embracing the high-value specialty segments via

arresting proliferation [95].

122 Plants for the Future

tion by their targets [101].

**4. Conclusions and future perspectives**

platform intermediates needed for bulk production.

biomass yield.

applications.

Knowledge of plant biosynthetic pathways will also provide valuable opportunities for metabolic engineering, as well as access to chemical transformations unique to plants. It is expected that genomics will corroborate plant biochemistry as researchers seek to under‐ stand the metabolic pathways for the synthesis of high value-added products. Identifying rate-limiting steps in their synthesis could provide targets for genetically engineered biochemical pathways to produce augmented amounts of useful compounds and new compounds in plant cells. Although, plant-based production of novel compounds such as biopolymers (e.g., polyhydroxyalkanoates) in plant tissues has been documented [105], plant metabolic pathways are complex and often feature multiple levels of regulation suggest‐ ing that it will be difficult to target the best intervention points and accurately predict the outcome [106]. However, recent developments in knowledge-based metabolic engineering strategies, that is the storing and mining of genomic, transcriptomic, proteomic, and metabolomic data, might permit to generate models of metabolic pathways useful to define and refine optimal intervention strategies for synthesis of specific chemicals for industrial applications [107].

The recent emergence of synthetic biology as the basis for metabolic engineering in plants promises to positively change the way in which different intervention strategies are selected and implemented, as is already the case in microbes [108,109]. In this context, metabolic engineering has contributed successfully in the last two decades towards broadening the product portfolio containing various value-added and commodity chemicals and materi‐ als from renewable resources [108,110]. Obviously, the attempts of engineering plants using the strategies employed for microbes are definitively more cumbersome. However, plant scientists have already established efficient methods for the routine genetic transforma‐ tion of the majority of our principal crops. Using these technologies, they have also achieved substantial progress in understanding and manipulating plant primary and secondary metabolism. Moreover, further research on metabolic regulation and genetic information and on novel tools for genetic modifications may help to overcome the limitations for breeding varieties that meet targeted applications and end-uses. This will certainly help maximize their value throughout the whole bio-based value chain for biorefining applica‐ tions or eventually breeding varieties with in-built new traits that are creating higher valueadded products.
