Laser Microdissection: A High-Precision Approach to Isolate Specific Cell Types from Any Plant Species for Downstream Molecular Analyses.

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.

Transcriptome analysis of the pulp of citrus fruitlets suggests that domestication enhanced growth processes and reduced chemical defenses increasing palatability.

To identify key traits brought about by citrus domestication, we have analyzed the transcriptomes of the pulp of developing fruitlets of inedible wild Ichang papeda (Citrus ichangensis), acidic Sun Chu Sha Kat mandarin (C. reticulata) and three palatable segregants of a cross between commercial Clementine (C. x clementina) and W. Murcott (C. x reticulata) mandarins, two pummelo/mandarin admixtures of worldwide distribution. RNA-seq comparison between the wild citrus and the ancestral sour mandarin identified 7267 differentially expressed genes, out of which 2342 were mapped to 117 KEGG pathways. From the remaining genes, a set of 2832 genes was functionally annotated and grouped into 45 user-defined categories. The data suggest that domestication promoted fundamental growth processes to the detriment of the production of chemical defenses, namely, alkaloids, terpenoids, phenylpropanoids, flavonoids, glucosinolates and cyanogenic glucosides. In the papeda, the generation of energy to support a more active secondary metabolism appears to be dependent upon upregulation of glycolysis, fatty acid degradation, Calvin cycle, oxidative phosphorylation, and ATP-citrate lyase and GABA pathways. In the acidic mandarin, downregulation of cytosolic citrate degradation was concomitant with vacuolar citrate accumulation. These changes affected nitrogen and carbon allocation in both species leading to major differences in organoleptic properties since the reduction of unpleasant secondary metabolites increases palatability while acidity reduces acceptability. The comparison between the segregants and the acidic mandarin identified 357 transcripts characterized by the occurrence in the three segregants of additional downregulation of secondary metabolites and basic structural cell wall components. The segregants also showed upregulation of genes involved in the synthesis of methyl anthranilate and furaneol, key substances of pleasant fruity aroma and flavor, and of sugar transporters relevant for sugar accumulation. Transcriptome and qPCR analysis in developing and ripe fruit of a set of genes previously associated with citric acid accumulation, demonstrated that lower acidity is linked to downregulation of these regulatory genes in the segregants. The results suggest that the transition of inedible papeda to sour mandarin implicated drastic gene expression reprograming of pivotal pathways of the primary and secondary metabolism, while palatable mandarins evolved through progressive refining of palatability properties, especially acidity.

IDA (INFLORESCENCE DEFICIENT IN ABSCISSION)-like peptides and HAE (HAESA)-like receptors regulate corolla abscission in Nicotiana benthamiana flowers.

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.

Differential expression of IDA (INFLORESCENCE DEFICIENT IN ABSCISSION)-like genes in Nicotiana benthamiana during corolla abscission, stem growth and water stress.

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.

Genomics of the origin and evolution of Citrus.

The genus Citrus, comprising some of the most widely cultivated fruit crops worldwide, includes an uncertain number of species. Here we describe ten natural citrus species, using genomic, phylogenetic and biogeographic analyses of 60 accessions representing diverse citrus germ plasms, and propose that citrus diversified during the late Miocene epoch through a rapid southeast Asian radiation that correlates with a marked weakening of the monsoons. A second radiation enabled by migration across the Wallace line gave rise to the Australian limes in the early Pliocene epoch. Further identification and analyses of hybrids and admixed genomes provides insights into the genealogy of major commercial cultivars of citrus. Among mandarins and sweet orange, we find an extensive network of relatedness that illuminates the domestication of these groups. Widespread pummelo admixture among these mandarins and its correlation with fruit size and acidity suggests a plausible role of pummelo introgression in the selection of palatable mandarins. This work provides a new evolutionary framework for the genus Citrus.

Corrigendum: Cell Wall Remodeling in Abscission Zone Cells during Ethylene-Promoted Fruit Abscission in Citrus.

[This corrects the article on p. 126 in vol. 8, PMID: 28228766.].

Cell Wall Remodeling in Abscission Zone Cells during Ethylene-Promoted Fruit Abscission in Citrus.

Abscission is a cell separation process by which plants can shed organs such as fruits, leaves, or flowers. The process takes place in specific locations termed abscission zones. In fruit crops like citrus, fruit abscission represents a high percentage of annual yield losses. Thus, understanding the molecular regulation of abscission is of capital relevance to control production. To identify genes preferentially expressed within the citrus fruit abscission zone (AZ-C), we performed a comparative transcriptomics assay at the cell type resolution level between the AZ-C and adjacent fruit rind cells (non-abscising tissue) during ethylene-promoted abscission. Our strategy combined laser microdissection with microarray analysis. Cell wall modification-related gene families displayed prominent representation in the AZ-C. Phylogenetic analyses of such gene families revealed a link between phylogenetic proximity and expression pattern during abscission suggesting highly conserved roles for specific members of these families in abscission. Our transcriptomic data was validated with (and strongly supported by) a parallel approach consisting on anatomical, histochemical and biochemical analyses on the AZ-C during fruit abscission. Our work identifies genes potentially involved in organ abscission and provides relevant data for future biotechnology approaches aimed at controlling such crucial process for citrus yield.

The IDA Peptide Controls Abscission in Arabidopsis and Citrus.

Organ abscission is an important process in plant development and reproduction. During abscission, changes in cellular adhesion of specialized abscission zone cells ensure the detachment of infected organs or those no longer serving a function to the plant. In addition, abscission also plays an important role in the release of ripe fruits. Different plant species display distinct patterns and timing of organ shedding, most likely adapted during evolution to their diverse life styles. However, it appears that key regulators of cell separation may have conserved function in different plant species. Here, we investigate the functional conservation of the citrus ortholog of the Arabidopsis peptide ligand INFLORESCENCE DEFICIENT IN ABSCISSION (AtIDA), controlling floral organ abscission. We discuss the possible implications of modifying the citrus IDA ortholog for citrus fruit production.

Novel insights into the Citrus sinensis nonhost response suggest photosynthesis decline, abiotic stress networks and secondary metabolism modifications.

Plants are constantly exposed to stress factors. Biotic stress is produced by living organisms such as pathogens, whereas abiotic stress by unfavourable environmental conditions. In Citrus species, one of the most important fruit crops in the world, these stresses generate serious limitations in productivity. Through biochemical and transcriptomic assays, we had previously characterised the Citrus sinensis (L.) Osbeck nonhost response to Xanthomonas campestris pv. vesicatoria (Doidge), in contrast to Asiatic citrus canker infection caused by Xanthomonas citri subsp. citri (Hasse). A hypersensitive response (HR) including changes in the expression of several transcription factors was reported. Here, a new exhaustive analysis of the Citrus sinensis transcriptomes previously obtained was performed, allowing us to detect the over-representation of photosynthesis, abiotic stress and secondary metabolism processes during the nonhost HR. The broad downregulation of photosynthesis-related genes was correlated with an altered photosynthesis physiology. The high number of heat shock proteins and genes related to abiotic stress, including aquaporins, suggests that stresses crosstalk. Additionally, the secondary metabolism exhibited lignin and carotenoid biosynthesis modifications and expression changes in the cell rescue GSTs. In conclusion, novel features of the Citrus nonhost HR, an important part of the plants' defence against disease that has yet to be fully exploited in plant breeding programs, are presented.

Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domestication.

Cultivated citrus are selections from, or hybrids of, wild progenitor species whose identities and contributions to citrus domestication remain controversial. Here we sequence and compare citrus genomes--a high-quality reference haploid clementine genome and mandarin, pummelo, sweet-orange and sour-orange genomes--and show that cultivated types derive from two progenitor species. Although cultivated pummelos represent selections from one progenitor species, Citrus maxima, cultivated mandarins are introgressions of C. maxima into the ancestral mandarin species Citrus reticulata. The most widely cultivated citrus, sweet orange, is the offspring of previously admixed individuals, but sour orange is an F1 hybrid of pure C. maxima and C. reticulata parents, thus implying that wild mandarins were part of the early breeding germplasm. A Chinese wild 'mandarin' diverges substantially from C. reticulata, thus suggesting the possibility of other unrecognized wild citrus species. Understanding citrus phylogeny through genome analysis clarifies taxonomic relationships and facilitates sequence-directed genetic improvement.