Gregorio Hueros

Camelina sativa (L.) Crantz, a native European oilseed crop of the Brassicaceae family, is notable for its short life cycle, making it well-suited for crop rotation and diversification. Its seeds contain a high content of oil (36–47%) that is rich in polyunsaturated fatty acids (PUFAs) such as alpha-linolenic acid (ALA, C18:3, Ω-3) and linoleic acid (LA, C18:2, Ω-6). This oil has diverse industrial applications, including low-emission biofuels, animal feed, pharmaceuticals, biolubricants, bioplastics, and cosmetics. We analyzed the expression of seven key enzymes involved in fatty acid biosynthesis across nine C. sativa accessions at three stages of silique development using highly efficient qRT-PCR assays designed for the target genes and a normalizing control. Our detailed expression analysis revealed significant variation across varieties, with only the gene FAB2c exhibiting genotype-independent expression, indicating a constitutive and essential role in monounsaturated fatty acid (MUFA) biosynthesis. Other genes showed significant interactions between the variety and developmental stage, highlighting the combined influences of genetic background and silique maturation on gene regulation. V18 emerges as particularly promising, exhibiting elevated expression of genes linked to PUFA and VLCFA biosynthesis—traits of significance for food, biofuel, and industrial applications. These findings, together with the developed qRT-PCR assays, provide valuable tools for selecting Camelina varieties with optimized genetic profiles, highlighting the potential of harnessing natural transcriptional diversity for crop improvement.

ZmTCRR1 and 2 are type-A response regulators expressed in the maize endosperm transfer cells (TC). While type-B response regulators transcriptionally control canonical type-A response regulators, as part of the cytokinin signal transduction mechanism, the ZmTCRRs are regulated by ZmMRP1, a master regulator of TC identity. In addition, the corresponding proteins are not detected in the TC, accumulating in the inner endosperm cells instead. These features suggest these molecules are not involved in classical, cell-autonomous, cytokinin signalling pathways. Using transgenic Arabidopsis plants ectopically expressing these genes, we have shown that ZmTCRR1 and 2 can modulate auxin and cytokinin signalling, respectively. In Arabidopsis, the ectopic expression of ZmTCRR2 blocked, almost completely, cytokinin perception. Given the conservation of these signalling pathways at the molecular level, our results suggest that the ZmTCRRs modulate cytokinin and auxin perception in the inner endosperm cells.

Main conclusion Studies in cell wall bound invertase mutants indicate that the promoter of the transfer cell-specific transcription factor, ZmMRP - 1 , is modulated by the carbohydrate balance. Transfer cells are highly specialized plant cells located at the surfaces that need to support an intensive exchange of nutrients, such as the entrance of fruits, seeds and nodules or the young branching points along the stem. ZmMRP-1 is a one-domain MYB-related transcription factor specifically expressed at the transfer cell layer of the maize endosperm. Previous studies demonstrated that this factor regulates the expression of a large number of transfer cell-specific genes, and suggested that ZmMRP-1 is a key regulator of the differentiation of this tissue. The expression of this gene is largely dominated by positional cues, but within the ZmMRP-1 expressing cells the promoter appears to be modulated by sugars. Here we have investigated in vivo this modulation. Using maize and Arabidopsis mutants for cell wall invertase genes, we found that the absence of cell wall invertase activity is a major inductive signal of the ZmMRP-1 expression.

Two subtilisin-like proteases show highly specific and complementary expression patterns in developing grains. These genes label the complete surface of the filial–maternal interface, suggesting a role in filial epithelial differentiation. The cereal endosperm is the most important source of nutrition and raw materials for mankind, as well as the storage compartment enabling initial growth of the germinating plantlets. The development of the different cell types in this tissue is regulated environmentally, genetically and epigenetically, resulting in the formation of top–bottom, adaxial–abaxial and surface–central axes. However, the mechanisms governing the interactions among the different inputs are mostly unknown. We have screened a kernel cDNA library for tissue-specific transcripts as initial step to identify genes relevant in cell differentiation. We report here on the isolation of two maize subtilisin-related genes that show grain-specific, surficial expression. zmsbt1 (Zea mays Subtilisin1) is expressed at the developing aleurone in a time-regulated manner, while zmsbt2 concentrates at the pedicel in front of the endosperm basal transfer layer. We have shown that their presence, early in the maize caryopsis development, is dependent on proper initial tissue determination, and have isolated their promoters to produce transgenic reporter lines that assist in the study of their regulation.

Mutant collections are an invaluable source of material on which forward genetic approaches allow the identification of genes affecting a wide variety of biological processes. However, some particular developmental stages and morphological structures may resist analysis due to their physical inaccessibility or to deleterious effects associated to their modification. Furthermore, lethal mutations acting early in development may escape detection. We have approached the characterisation of 101 maize seed mutants, selected from a collection of 27500 visually screened Mu-insertion lines, using a molecular marker approach based on a set of genes previously ascribed to different tissue compartments within the early developing kernel. A streamlined combination of qRT-PCR assays has allowed us to preliminary pinpoint the affected compartment, establish developmental comparisons to WT siblings and select mutant lines with alterations in the different compartments. Furthermore, clusters of markers co-affected by the underlying mutation were identified. We have analysed more extensively a set of lines presenting significant variation in transfer cell-associated expression markers, and have performed morphological observations, and immunolocalization experiments to confirm the results, validating this approach as an efficient mutant description tool.

Background: Two component systems (TCS) are phosphotransfer-based signal transduction pathways first discovered in bacteria, where they perform most of the sensing tasks. They present a highly modular structure, comprising a receptor with histidine kinase activity and a response regulator which regulates gene expression or interacts with other cell components. A more complex framework is usually found in plants and fungi, in which a third component transfers the phosphate group from the receptor to the response regulator. They play a central role in cytokinin mediated functions in plants, affecting processes such as meristem growth, phyllotaxy, seed development, leaf senescence or tissue differentiation. We have previously reported the expression and cellular localization of a type A response regulator, ZmTCRR-1, in the transfer cells of the maize seed, a tissue critical for seed filling and development, and described its regulation by a tissue specific transcription factor. In this work we investigate the expression and localization of other components of the TCS signalling routes in the maize seed and initiate the characterization of their interactions. Results: The discovery of a new type A response regulator, ZmTCRR-2, specifically expressed in the transfer cells and controlled by a tissue specific transcription factor suggests a previously unknown role for TCS in the biology of transfer cells. We have characterized other canonical TCS molecules, including 6 histidine kinases and 3 phosphotransfer proteins, potentially involved in the atypical transduction pathway defined by ZmTCRR-1 and 2. We have identified potential upstream interactors for both proteins and shown that they both move into the developing endosperm. Furthermore, ZmTCRR-1 expression in an heterologous system (Arabidopsis thaliana) is directed to xylem parenchyma cells, probably involved in transport processes, one of the major roles attributed to the transfer cell layer. Conclusions: Our data prove the expression of the effector elements of a TCS route operating in the transfer cells under developmental control. Its possible role in integrating external signals with seed developmental processes is discussed.

ZmMRP-1 is a single MYB-domain transcription factor specifically expressed in the transfer cell layer of the maize endosperm, where it directly regulates the expression of a number of transfer cell specific genes and very likely contributes to the regulation of the transfer cell differentiation process. It is still a matter of debate, however, how this type of transcription factors interact with the promoter sequences they regulate. In this work we have investigated the existence of proteins interacting with ZmMRP-1 in the transfer cell nuclei. In a yeast double-hybrid screen we identified two related maize proteins, ZmMRPI-1 and ZmMRPI-2 belonging to the C₂H₂ zinc finger protein family, which interact with ZmMRP-1 and modulate its activity on transfer cell specific promoters. Two ZmMRPI orthologous genes were also identified in the rice and Arabidopsis genomes. The expression pattern in maize and Arabidopsis suggest a role for these proteins in gene regulation at the exchange surfaces where ZmMRP-1 is expressed providing the first indication of their function. We show that this previously uncharacterized family of proteins encodes nuclear proteins that interact with MYB-related transcription factors through their C-terminal conserved domain.

Transfer cells are highly modified plant cells specialized in the transport of solutes. They differentiate at many plant exchange surfaces, including phloem loading and unloading zones such as those present in the sink organs and seeds. In maize (Zea mays) seeds, transfer cells are located at the base of the endosperm. It is currently unknown how apical-basal polarity is established or why the peripheral cells at the base of the endosperm differentiate into transfer instead of aleurone cells. Here, we show that in epidermal cells committed to develop into aleurone cells, the ectopic expression of the transfer cell-specific transcriptional activator Myb-Related Protein-1 (MRP-1) is sufficient to temporarily transform them into transfer cells. These transformed cells acquire distinct transfer cell features, such as cell wall ingrowths and an elongated shape. In addition, they express a number of MRP-1 target genes presumably involved in defense. We also show that the expression of MRP-1 is needed to maintain the transfer cell phenotype. Later in development, an observed reduction in the ectopic expression of MRP-1 was followed by the reversion of the transformed cells, which then acquire aleurone cell features.

Transfer cells have specializations that facilitate the transport of solutes across plant exchange surfaces. ZmMRP-1 is a maize (Zea mays) endosperm transfer cell-specific transcriptional activator that plays a central role in the regulatory pathways controlling transfer cell differentiation and function. The present work investigates the signals controlling the expression of ZmMRP-1 through the production of transgenic lines of maize, Arabidopsis, tobacco and barley containing ZmMRP-1promoter:GUS reporter constructs. The GUS signal predominantly appeared in regions of active transport between source and sink tissues, including nematode-induced feeding structures and at sites of vascular connection between developing organs and the main plant vasculature. In those cases, promoter induction was associated with the initial developmental stages of transport structures. Significantly, transfer cells also differentiated in these regions suggesting that, independent of species, location or morphological features, transfer cells might differentiate in a similar way under the influence of conserved induction signals. In planta and yeast experiments showed that the promoter activity is modulated by carbohydrates, glucose being the most effective inducer.