Francisco R. Tadeo

Virus-induced gene silencing (VIGS) is a powerful and cost-effective biotechnological tool for studying gene function in plants. It allows for rapid downregulation of gene expression without the need for tissue culture or genetic transformation. While VIGS is transient and may pose challenges for certain developmental genes, it has been successfully used to study organ abscission. Using a VIGS protocol based on Citrus leaf blotch virus (CLBV) viral vectors, we were able to characterize the regulatory role of INFLORESCENCE DEFICIENT IN ABSCISSION (IDA)-like genes and their potential HAESA (HAE)-like receptors in the process of corolla abscission in Nicotiana benthamiana flowers.

The function of abscission zones (AZs) determines the timing of fleshy fruit abscission, with important consequences not only for the optimal fruit harvest, but also on the overall final fruit quality. In this context, chemical treatments are commonly used at different stages of fruit development to control fruit abscission, which can also have positive or negative effects on fruit quality. In the current review, we examine commonly used chemicals that affect the metabolic activity in the AZs of fleshy fruit, in addition to their effects on fruit quality characteristics. The main hormone metabolism and signaling in the AZ include that of ethylene, auxin, abscisic acid and jasmonates, and the molecular components that are involved are covered and discussed, in addition to how these hormones work together to regulate AZ activity and hence, affect fruit quality. We focus on studies that have provided new insight into possible protein complexes that function in the AZ, including multiple MADS-box transcription factors, with potential overlapping regulatory roles which exist between AZ development, ethylene production, AZ activation, fruit ripening and overall fruit quality. The view of the AZ as a cross roads where multiple pathways and signals are integrated is discussed.

Plants display a great diversity of particular cell types that obviously perform functions and regulations that are essential for successful growth and development, whether under optimal or adverse conditions. The functions performed by each of these particular cell types must be associated with specific transcriptomic, proteomic, and metabolic profiles that cannot be disentangled by analyzing whole plant organs and tissues. Laser microdissection is a technique for the collection of specific cell types in plant organs and tissues comprising heterogeneous cell populations. It has been successfully used for physiological and molecular studies. Laser microdissection can be applied to any plant species as long as it is possible to reliably identify the cell types of interest. Here, we describe step by step, using citrus as a model plant, a fast, simple, easy to perform, and experimentally validated protocol to collect cells from the abscission zone, a specific tissue that is difficult to access and whose activity is important in the response of plants to adverse environmental conditions. Key words Abscission Calyx abscission zone Cell separation Citrus Cryosectioning Ethylene Laminar abscission zone

Background: Abscission is an active, organized, and highly coordinated cell separation process enabling the detachment of aerial organs through the modification of cell-to-cell adhesion and breakdown of cell walls at specific sites on the plant body known as abscission zones. In Arabidopsis thaliana, abscission of floral organs and cauline leaves is regulated by the interaction of the hormonal peptide INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), a pair of redundant receptor-like protein kinases, HAESA (HAE) and HAESA-LIKE2 (HSL2), and SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) co-receptors. However, the functionality of this abscission signaling module has not yet been demonstrated in other plant species. Results: The expression of the pair of NbenIDA1 homeologs and the receptor NbenHAE.1 was supressed at the base of the corolla tube by the inoculation of two virus-induced gene silencing (VIGS) constructs in Nicotiana benthamiana. These gene suppression events arrested corolla abscission but did not produce any obvious effect on plant growth. VIGS plants retained a higher number of corollas attached to the flowers than control plants, an observation related to a greater corolla breakstrength. The arrest of corolla abscission was associated with the preservation of the parenchyma tissue at the base of the corolla tube that, in contrast, was virtually collapsed in normal corollas. In contrast, the inoculation of a viral vector construct that increased the expression of NbenIDA1A at the base of the corolla tube negatively affected the growth of the inoculated plants accelerating the timing of both corolla senescence and abscission. However, the heterologous ectopic overexpression of citrus CitIDA3 and Arabidopsis AtIDA in N. benthamiana did not alter the standard plant phenotype suggesting that the proteolytic processing machinery was unable to yield active peptides. Conclusion: Here, we demonstrate that the pair of NbenIDA1 homeologs encoding small peptides of the IDA-like family and the receptor NbenHAE.1 control cellular breakdown at the base of the corolla tube awhere an adventitious AZ should be formed and, therefore, corolla abscission in N. benthamiana flowers. Altogether, our results provide the first evidence supporting the notion that the IDA-HAE/HSL2 signaling module is conserved in angiosperms.

Orange fruit abscission usually occurs at the final stage of fruit maturation but in some areas of citrus production, in advance of the usual harvest period, and sometimes suddenly and intensely. The reasons for this precocious citrus fruit abscission remains unclear. Therefore, the aim of this study was to try to clarify what the determinants of this phenomenon are. A multi-site experimentation was carried out on six orange cultivars, in Corsica, Spain and Tunisia where the phenomenon of early massive fruit drop varies. Climatic parameters, fruit maturity parameters and fruit detachment force (FDF) were recorded along the fruit maturation period. Respectively to the fruit drop, the FDF decreased in Tunisia and in Spain until the fruit falls, whereas in Corsica, it remained relatively constant throughout fruit maturation. Although data on fruit maturity parameters (rind color, acidity and total soluble solids) differed at the three sites, their evolution was similar during the period of maturation. FDF was not related to changes in any fruit maturity parameters, and more likely depended on changes in temperatures on days when the mean temperature was above 13 °C. Massive fruit abscission could be linked to the earlier more rapid restart of vegetative growth in Tunisia and Spain than in Corsica.

Background: IDA (INFLORESCENCE DEFICIENT IN ABSCISSION)-like signaling peptides and the associated HAE (HAESA)-like family of receptor kinases were originally reported in the model plantArabidopsis thaliana(Arabidopsis) to be deeply involved in the regulation of abscission. Actually, IDA peptides, as cell-to-cell communication elements, appear to be implicated in many developmental processes that rely on cell separation events, and even in the responses to abiotic stresses. However, the knowledge related to the molecular machinery regulating abscission in economically important crops is scarce. In this work, we determined the conservation and phylogeny of theIDA-like andHAE-like gene families in relevant species of the Solanaceae family and analyzed the expression of these genes in the allopolyploidNicotiana benthamiana,in order to identify members involved in abscission, stem growth and in the response to drought conditions.Results: The phylogenetic relationships among theIDA-like members of the Solanaceae studied, grouped the two pairs of NbenIDA1 and NbenIDA2 protein homeologs with the Arabidopsis prepropeptides related to abscission. Analysis of promoter regions searching for regulatory elements showed that these two pairs of homeologs contained both hormonal and drought response elements, althoughNbenIDA2Alacked the hormonal regulatory elements. Expression analyses showed that the pair ofNbenIDA1homeologs were upregulated during corolla abscission.NbenIDA1andNbenIDA2pairs showed tissue differential expression under water stress conditions, sinceNbenIDA1homeologs were highly expressed in stressed leaves whileNbenIDA2homeologs, especiallyNbenIDA2B, were highly expressed in stressed roots. In non-stressed active growing plants, nodes and internodes were the tissues with the highest expression levels of all members of theIDA-like family and their putativeHAE-like receptors.Conclusion: Our results suggest that the pair ofNbenIDA1homeologs are involved in the natural process of corolla abscission while both pairs ofNbenIDA1andNbenIDA2homeologs are implicated in the response to water stress. The data also suggest that IDA peptides may be important during stem growth and development. These results provide additional evidence that the functional module formed by IDA peptides and its receptor kinases, as defined in Arabidopsis, may also be conserved in Solanaceae.

Background IDA (INFLORESCENCE DEFICIENT IN ABSCISSION)-like signaling peptides and the associated HAE (HAESA)-like family of receptor kinases were originally reported in the model plant Arabidopsis thaliana (Arabidopsis) to be deeply involved in the regulation of abscission. Actually, IDA peptides, as cell-to-cell communication elements, appear to be implicated in many developmental processes that rely on cell separation events, and even in the responses to abiotic stresses. However, the knowledge related to the molecular machinery regulating abscission in economically important crops is scarce. In this work, we determined the conservation and phylogeny of the IDA -like and HAE -like gene families in relevant species of the Solanaceae family and analyzed the expression of these genes in the allopolyploid Nicotiana benthamiana, in order to identify members involved in abscission, stem growth and in the response to drought conditions. Results The phylogenetic relationships among the IDA -like members of the Solanaceae studied, grouped the two pairs of NbenIDA1 and NbenIDA2 protein homeologs with the Arabidopsis prepropeptides related to abscission. Analysis of promoter regions searching for regulatory elements showed that these two pairs of homeologs contained both hormonal and drought response elements, although NbenIDA2A lacked the hormonal regulatory elements. Expression analyses showed that the pair of NbenIDA1 homeologs were upregulated during corolla abscission. NbenIDA1 and NbenIDA2 pairs showed tissue differential expression under water stress conditions, since NbenIDA1 homeologs were highly expressed in stressed leaves while NbenIDA2 homeologs, especially NbenIDA2B , were highly expressed in stressed roots. In non-stressed active growing plants, nodes and internodes were the tissues with the highest expression levels of all members of the IDA -like family and their putative HAE -like receptors. Conclusion Our results suggest that the pair of NbenIDA1 homeologs are involved in the natural process of corolla abscission while both pairs of NbenIDA1 and NbenIDA2 homeologs are implicated in the response to water stress. The data also suggest that IDA peptides may be important during stem growth and development. These results provide additional evidence that the functional module formed by IDA peptides and its receptor kinases, as defined in Arabidopsis, may also be conserved in Solanaceae.

Background IDA (INFLORESCENCE DEFICIENT IN ABSCISSION)-like signaling peptides and the associated HAE (HAESA)-like family of receptor kinases were originally reported in the model plant Arabidopsis thaliana (Arabidopsis) to be deeply involved in the regulation of abscission. Actually, IDA peptides, as cell-to-cell communication elements, appear to be implicated in many developmental processes that rely on cell separation events, and even in the responses to abiotic stresses. However, the knowledge related to the molecular machinery regulating abscission in economically important crops is scarce. In this work, we determined the conservation and phylogeny of the IDA -like and HAE -like gene families in relevant species of the Solanaceae family and analyzed the expression of these genes in the allopolyploid Nicotiana benthamiana, in order to identify members involved in abscission, stem growth and in the response to drought conditions. Results The phylogenetic relationships among the IDA -like members of the Solanaceae studied, grouped the two pairs of NbenIDA1 and NbenIDA2 protein homeologs with the Arabidopsis prepropeptides related to abscission. Analysis of promoter regions searching for regulatory elements showed that these two pairs of homeologs contained both hormonal and drought response elements, although NbenIDA2A lacked the hormonal regulatory elements. Expression analyses showed that the pair of NbenIDA1 homeologs were upregulated during corolla abscission. NbenIDA1 and NbenIDA2 pairs showed tissue differential expression under water stress conditions, since NbenIDA1 homeologs were highly expressed in stressed leaves while NbenIDA2 homeologs, especially NbenIDA2B , were highly expressed in stressed roots. In non-stressed active growing plants, nodes and internodes were the tissues with the highest expression levels of all members of the IDA -like family and their putative HAE -like receptors. Conclusion Our results suggest that the pair of NbenIDA1 homeologs are involved in the natural process of corolla abscission while both pairs of NbenIDA1 and NbenIDA2 homeologs are implicated in the response to water stress. The data also suggest that IDA peptides may be important during stem growth and development. These results provide additional evidence that the functional module formed by IDA peptides and its receptor kinases, as defined in Arabidopsis, may also be conserved in Solanaceae.

Background: Small RNAs regulate a wide variety of processes in plants, from organ development to both biotic and abiotic stress response. Being master regulators in genetic networks, their biogenesis and action is a fundamental aspect to characterize in order to understand plant growth and development. Three main gene families are critical components of RNA silencing: DICER-LIKE (DCL), ARGONAUTE (AGO) and RNA-DEPENDENT RNA POLYMERASE (RDR). Even though they have been characterized in other plant species, there is no information about these gene families in Citrus sinensis, one of the most important fruit species from both economical and nutritional reasons. While small RNAs have been implicated in the regulation of multiple aspects of plant growth and development, their role in the abscission process has not been characterized yet. Results: Using genome-wide analysis and a phylogenetic approach, we identified a total of 13 AGO, 5 DCL and 7 RDR genes. We characterized their expression patterns in root, leaf, flesh, peel and embryo samples using RNA-seq data. Moreover, we studied their role in fruit abscission through gene expression analysis in fruit rind compared to abscission zone from samples obtained by laser capture microdissection. Interestingly, we determined that the expression of several RNA silencing factors are down-regulated in fruit abscission zone, being particularly represented gene components of the RNA-dependent DNA Methylation pathway, indicating that repression of this process is necessary for fruit abscission to take place in Citrus sinensis. Conclusions: The members of these 3 families present characteristic conserved domains and distinct expression patterns. We provide a detailed analysis of the members of these families and improved the annotation of some of these genes based on RNA-seq data. Our data suggests that the RNA-dependent DNA Methylation pathway is involved in the important fruit abscission process in C. sinensis.

Background IDA (INFLORESCENCE DEFICIENT IN ABSCISSION)-like signaling peptides and the associated HAE (HAESA)-like family of receptor kinases were originally reported in the model plant Arabidopsis thaliana (Arabidopsis) to be involved in the regulation of abscission. IDA peptides, as cell-to-cell communication elements, appear to be implicated in many developmental processes that rely on cell separation events, and even in the responses to abiotic stresses. However, the knowledge related to the molecular machinery regulating abscission and other cell separation processes in economically important crops is scarce. In this work, we determined the conservation and phylogeny of the IDA-like and HAE-like gene families in relevant species of the Solanaceae family and analyzed the expression of these genes in Nicotiana benthamiana to identify members involved in abscission, stem growth and response to drought conditions. Results The phylogenetic relationships among the IDA-like members of the species of Solanaceae studied, grouped the NbenIDA1 (A and B) and NbenIDA2 (A and B) pairs of genes with the Arabidopsis prepropeptides related to abscission. In silico analyses of the regulatory elements of the promoter region of IDA-like members showed that these two pairs of genes, NbenIDA1 and NbenIDA2, contained drought and hormonal response elements, although NbenIDA2A lacked the hormonal regulatory elements. Expression analyses showed that both NbenIDA1 genes were upregulated during corolla abscission. NbenIDA1 and NbenIDA2 genes showed tissue differential expression under water stress conditions, since NbenIDA1 genes were highly expressed in stressed leaves while NbenIDA2 genes, especially NbenIDA2B, were highly expressed in stressed roots. In non-stressed active growing plants, nodes and internodes were the tissues with the highest expression levels of all members of the IDA-like family and their putative HAE-like receptors. Conclusion The results suggest that NbenIDA1A and NbenIDA1B are both involved in the natural process of corolla abscission while the NbenIDA1 and NbenIDA2 pair of genes are implicated in the response to water stress. The data also suggest that IDA peptides may be important during stem growth and development. These results add new evidence that the functional module formed by IDA peptides and its receptor kinases, as defined in Arabidopsis, may also be conserved in Solanaceae.