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Abstract Background The outer cell wall of the pollen grain (exine) is an extremely resistant structure containing sporopollenin, a mixed polymer made up of fatty acids and phenolic compounds. The synthesis of sporopollenin in the tapetal cells and its proper deposition on the pollen surface are essential for the development of viable pollen. The beginning of microsporogenesis and pollen maturation in perennial plants from temperate climates, such as peach, is conditioned by the duration of flower bud dormancy. In order to identify putative genes involved in these processes, we analyzed the results of previous genomic experiments studying the dormancy-dependent gene expression in different peach cultivars. Results The expression of 50 genes induced in flower buds after the endodormancy period (flower-bud late genes) was compared in ten cultivars of peach with different dormancy behaviour. We found two co-expression clusters enriched in putative orthologs of sporopollenin synthesis and deposition factors in Arabidopsis. Flower-bud late genes were transiently expressed in anthers coincidently with microsporogenesis and pollen maturation processes. We postulated the participation of some flower-bud late genes in the sporopollenin synthesis pathway and the transcriptional regulation of late anther development in peach. Conclusions Peach and the model plant Arabidopsis thaliana show multiple elements in common within the essential sporopollenin synthesis pathway and gene expression regulatory mechanisms affecting anther development. The transcriptomic analysis of dormancy-released flower buds proved to be an efficient procedure for the identification of anther and pollen development genes in perennial plants showing seasonal dormancy.

Abstract Background Reproductive biology in citrus is still poorly understood. Although in recent years several efforts have been made to study pollen-pistil interaction and self-incompatibility, little information is available about the molecular mechanisms regulating these processes. Here we report the identification of candidate genes involved in pollen-pistil interaction and self-incompatibility in clementine (Citrus clementina Hort. ex Tan.). These genes have been identified comparing the transcriptomes of laser-microdissected stylar canal cells (SCC) isolated from two genotypes differing for self-incompatibility response ('Comune', a self-incompatible cultivar and 'Monreal', a self- compatible mutation of 'Comune'). Results The transcriptome profiling of SCC indicated that the differential regulation of few specific, mostly uncharacterized transcripts is associated with the breakdown of self-incompatibility in 'Monreal'. Among them, a novel F-box gene showed a drastic up-regulation both in laser microdissected stylar canal cells and in self-pollinated whole styles with stigmas of 'Comune' in concomitance with the arrest of pollen tube growth. Moreover, we identify a non-characterized gene family as closely associated to the self-incompatibility genetic program activated in 'Comune'. Three different aspartic-acid rich (Asp-rich) protein genes, located in tandem in the clementine genome, were over-represented in the transcriptome of 'Comune'. These genes are tightly linked to a DELLA gene, previously found to be up-regulated in the self-incompatible genotype during pollen-pistil interaction. Conclusion The highly specific transcriptome survey of the stylar canal cells identified novel genes which have not been previously associated with self-pollen rejection in citrus and in other plant species. Bioinformatic and transcriptional analyses suggested that the mutation leading to self-compatibility in 'Monreal' affected the expression of non-homologous genes located in a restricted genome region. Also, we hypothesize that the Asp-rich protein genes may act as Ca2+ "entrapping" proteins, potentially regulating Ca2+ homeostasis during self-pollen recognition.

Abstract Background Abscission is the cell separation process by which plants are able to shed organs. It has a great impact on the yield of most crop plants. At the same time, the process itself also constitutes an excellent model to study cell separation processes, since it occurs in concrete areas known as abscission zones (AZs) which are composed of a specific cell type. However, molecular approaches are generally hampered by the limited area and cell number constituting the AZ. Therefore, detailed studies at the resolution of cell type are of great relevance in order to accurately describe the process and to identify potential candidate genes for biotechnological applications. Results Efficient protocols for the isolation of specific citrus cell types, namely laminar abscission zone (LAZ) and petiolar cortical (Pet) cells based on laser capture microdissection (LCM) and for RNA microextraction and amplification have been developed. A comparative transcriptome analysis between LAZ and Pet from citrus leaf explants subjected to an in-vitro 24 h ethylene treatment was performed utilising microarray hybridization and analysis. Our analyses of gene functional classes differentially represented in ethylene-treated LAZ revealed an activation program dominated by the expression of genes associated with protein synthesis, protein fate, cell type differentiation, development and transcription. The extensive repertoire of genes associated with cell wall biosynthesis and metabolism strongly suggests that LAZ layers activate both catabolic and anabolic wall modification pathways during the abscission program. In addition, over-representation of particular members of different transcription factor families suggests important roles for these genes in the differentiation of the effective cell separation layer within the many layers contained in the citrus LAZ. Preferential expression of stress-related and defensive genes in Pet reveals that this tissue is likely to be reprogrammed to prevent pathogen attacks and general abiotic stresses after organ shedding. Conclusion The LCM-based data generated in this survey represent the most accurate description of the main biological processes and genes involved in organ abscission in citrus. This study provides novel molecular insight into ethylene-promoted leaf abscission and identifies new putative target genes for characterization and manipulation of organ abscission in citrus.

Abstract Background Improvement of Citrus, the most economically important fruit crop in the world, is extremely slow and inherently costly because of the long-term nature of tree breeding and an unusual combination of reproductive characteristics. Aside from disease resistance, major commercial traits in Citrus are improved fruit quality, higher yield and tolerance to environmental stresses, especially salinity. Results A normalized full length and 9 standard cDNA libraries were generated, representing particular treatments and tissues from selected varieties (Citrus clementina and C. sinensis) and rootstocks (C. reshni, and C. sinenis × Poncirus trifoliata) differing in fruit quality, resistance to abscission, and tolerance to salinity. The goal of this work was to provide a large expressed sequence tag (EST) collection enriched with transcripts related to these well appreciated agronomical traits. Towards this end, more than 54000 ESTs derived from these libraries were analyzed and annotated. Assembly of 52626 useful sequences generated 15664 putative transcription units distributed in 7120 contigs, and 8544 singletons. BLAST annotation produced significant hits for more than 80% of the hypothetical transcription units and suggested that 647 of these might be Citrus specific unigenes. The unigene set, composed of ~13000 putative different transcripts, including more than 5000 novel Citrus genes, was assigned with putative functions based on similarity, GO annotations and protein domains Conclusion Comparative genomics with Arabidopsis revealed the presence of putative conserved orthologs and single copy genes in Citrus and also the occurrence of both gene duplication events and increased number of genes for specific pathways. In addition, phylogenetic analysis performed on the ammonium transporter family and glycosyl transferase family 20 suggested the existence of Citrus paralogs. Analysis of the Citrus gene space showed that the most important metabolic pathways known to affect fruit quality were represented in the unigene set. Overall, the similarity analyses indicated that the sequences of the genes belonging to these varieties and rootstocks were essentially identical, suggesting that the differential behaviour of these species cannot be attributed to major sequence divergences. This Citrus EST assembly contributes both crucial information to discover genes of agronomical interest and tools for genetic and genomic analyses, such as the development of new markers and microarrays.

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., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Perez-Roman, Borredá, Tadeo and Talon.)

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.

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.

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.

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.

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.

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.

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.

Pathogens interaction with a host plant starts a set of immune responses that result in complex changes in gene expression and plant physiology. Light is an important modulator of plant defense response and recent studies have evidenced the novel influence of this environmental stimulus in the virulence of several bacterial pathogens. Xanthomonas citri subsp. citri is the bacterium responsible for citrus canker disease, which affects most citrus cultivars. The ability of this bacterium to colonize host plants is influenced by bacterial blue-light sensing through a LOV-domain protein and disease symptoms are considerably altered upon deletion of this protein. In this work we aimed to unravel the role of this photoreceptor during the bacterial counteraction of plant immune responses leading to citrus canker development. We performed a transcriptomic analysis in Citrus sinensis leaves inoculated with the wild type X. citri subsp. citri and with a mutant strain lacking the LOV protein by a cDNA microarray and evaluated the differentially regulated genes corresponding to specific biological processes. A down-regulation of photosynthesis-related genes (together with a corresponding decrease in photosynthesis rates) was observed upon bacterial infection, this effect being more pronounced in plants infected with the lov-mutant bacterial strain. Infection with this strain was also accompanied with the up-regulation of several secondary metabolism- and defense response-related genes. Moreover, we found that relevant plant physiological alterations triggered by pathogen attack such as cell wall fortification and tissue disruption were amplified during the lov-mutant strain infection. These results suggest the participation of the LOV-domain protein from X. citri subsp. citri in the bacterial counteraction of host plant defense response, contributing in this way to disease development.

Plants, when exposed to certain pathogens, may display a form of genotype-independent resistance, known as non-host response. In this study, the response of Citrus sinensis (sweet orange) leaves to Xanthomonas campestris pv. vesicatoria (Xcv), a pepper and tomato pathogenic bacterium, was analyzed through biochemical assays and cDNA microarray hybridization and compared with Asiatic citrus canker infection caused by Xanthomonas citri subsp. citri. Citrus leaves exposed to the non-host bacterium Xcv showed hypersensitive response (HR) symptoms (cell death), a defense mechanism common in plants but poorly understood in citrus. The HR response was accompanied by differentially expressed genes that are associated with biotic stress and cell death. Moreover, 58 transcription factors (TFs) were differentially regulated by Xcv in citrus leaves, including 26 TFs from the stress-associated families AP2-EREBP, bZip, Myb and WRKY. Remarkably, in silico analysis of the distribution of expressed sequence tags revealed that 10 of the 58 TFs, belonging to C2C2-GATA, C2H2, CCAAT, HSF, NAC and WRKY gene families, were specifically over-represented in citrus stress cDNA libraries. This study identified candidate TF genes for the regulation of key steps during the citrus non-host HR. Furthermore, these TFs might be useful in future strategies of molecular breeding for citrus disease resistance., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Abscission consists in the detachment of entire vegetative and reproductive organs due to cell separation processes occurring at the abscission zones (AZs) at specific positions of the plant body. From an evolutionary point of view, abscission is a highly advantageous process resulting into fruit and seed dispersal as well as the shedding of no longer useful organs. In an agricultural context, however, abscission may become a major limiting factor for crop productivity. Domestication of major crops included the selection of plants that did not naturally shed ripe fruits or seeds. The understanding of abscission is of great importance to control seed and fruit production and to improve breeding and harvesting practices. Thus, advances made on model plants and crops are of major importance since they may provide potential candidate genes for further biotechnological applications. Here, we review the current knowledge of the physiological, genetic and genomic aspects related to abscission including the most recently disclosed putative regulators that appear to be implicated in the development and/or activation of the AZs., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Leaf abscission is a common response of plants to drought stress. Some species, such as citrus, have evolved a specific behaviour in this respect, keeping their leaves attached to the plant body during water stress until this is released by irrigation or rain. This study successfully reproduced this phenomenon under controlled conditions (24h of water stress followed by 24h of rehydration) and used it to construct a suppression subtractive hybridization cDNA library enriched in genes involved in the early stages of rehydration-promoted leaf abscission after water stress. Sequencing of the library yielded 314 unigenes, which were spotted onto nylon membranes. Membrane hybridization with petiole (Pet)- and laminar abscission zone (LAZ)-enriched RNA samples corresponding to early steps in leaf abscission revealed an almost exclusive preferential gene expression programme in the LAZ. The data identified major processes such as protein metabolism, cell-wall modification, signalling, control of transcription and vesicle production, and transport as the main biological processes activated in LAZs during the early steps of rehydration-promoted leaf abscission after water stress. Based on these findings, a model for the early steps of citrus leaf abscission is proposed. In addition, it is suggested that CitbHLH1, the putative citrus orthologue of Arabidopsis BIGPETAL, may play major roles in the control of abscission-related events in citrus abscission zones.

Most studies of the biochemical and regulatory pathways that are associated with, and control, fruit expansion and ripening are based on homogenized bulk tissues, and do not take into consideration the multiplicity of different cell types from which the analytes, be they transcripts, proteins or metabolites, are extracted. Consequently, potentially valuable spatial information is lost and the lower abundance cellular components that are expressed only in certain cell types can be diluted below the level of detection. In this study, laser microdissection (LMD) was used to isolate epidermal and subepidermal cells from green, expanding Citrus clementina fruit and their transcriptomes were compared using a 20k citrus cDNA microarray and quantitative real-time PCR. The results show striking differences in gene expression profiles between the two cell types, revealing specific metabolic pathways that can be related to their respective organelle composition and cell wall specialization. Microscopy provided additional evidence of tissue specialization that could be associated with the transcript profiles with distinct differences in organelle and metabolite accumulation. Subepidermis predominant genes are primarily involved in photosynthesis- and energy-related processes, as well as cell wall biosynthesis and restructuring. By contrast, the most epidermis predominant genes are related to the biosynthesis of the cuticle, flavonoids, and defence responses. Furthermore, the epidermis transcript profile showed a high proportion of genes with no known function, supporting the original hypothesis that analysis at the tissue/cell specific levels can promote gene discovery and lead to a better understanding of the specialized contribution of each tissue to fruit physiology.

Background: Abscission is the cell separation process by which plants are able to shed organs. It has a great impact on the yield of most crop plants. At the same time, the process itself also constitutes an excellent model to study cell separation processes, since it occurs in concrete areas known as abscission zones (AZs) which are composed of a specific cell type. However, molecular approaches are generally hampered by the limited area and cell number constituting the AZ. Therefore, detailed studies at the resolution of cell type are of great relevance in order to accurately describe the process and to identify potential candidate genes for biotechnological applications., Results: Efficient protocols for the isolation of specific citrus cell types, namely laminar abscission zone (LAZ) and petiolar cortical (Pet) cells based on laser capture microdissection (LCM) and for RNA microextraction and amplification have been developed. A comparative transcriptome analysis between LAZ and Pet from citrus leaf explants subjected to an in-vitro 24 h ethylene treatment was performed utilising microarray hybridization and analysis. Our analyses of gene functional classes differentially represented in ethylene-treated LAZ revealed an activation program dominated by the expression of genes associated with protein synthesis, protein fate, cell type differentiation, development and transcription. The extensive repertoire of genes associated with cell wall biosynthesis and metabolism strongly suggests that LAZ layers activate both catabolic and anabolic wall modification pathways during the abscission program. In addition, over-representation of particular members of different transcription factor families suggests important roles for these genes in the differentiation of the effective cell separation layer within the many layers contained in the citrus LAZ. Preferential expression of stress-related and defensive genes in Pet reveals that this tissue is likely to be reprogrammed to prevent pathogen attacks and general abiotic stresses after organ shedding., Conclusion: The LCM-based data generated in this survey represent the most accurate description of the main biological processes and genes involved in organ abscission in citrus. This study provides novel molecular insight into ethylene-promoted leaf abscission and identifies new putative target genes for characterization and manipulation of organ abscission in citrus.

The main objective of this work was to identify and classify genes involved in the process of leaf abscission in Clementina de Nules (Citrus clementina Hort. Ex Tan.). A 7 K unigene citrus cDNA microarray containing 12 K spots was used to characterize the transcriptome of the ethylene-induced abscission process in laminar abscission zone-enriched tissues and the petiole of debladed leaf explants. In these conditions, ethylene induced 100% leaf explant abscission in 72 h while, in air-treated samples, the abscission period started later and took 240 h. Gene expression monitored during the first 36 h of ethylene treatment showed that out of the 12 672 cDNA microarray probes, ethylene differentially induced 725 probes distributed as follows: 216 (29.8%) probes in the laminar abscission zone and 509 (70.2%) in the petiole. Functional MIPS classification and manual annotation of differentially expressed genes highlighted key processes regulating the activation and progress of the cell separation that brings about abscission. These included cell-wall modification, lipid transport, protein biosynthesis and degradation, and differential activation of signal transduction and transcription control pathways. Expression data associated with the petiole indicated the occurrence of a double defensive strategy mediated by the activation of a biochemical programme including scavenging ROS, defence and PR genes, and a physical response mostly based on lignin biosynthesis and deposition. This work identifies new genes probably involved in the onset and development of the leaf abscission process and suggests a different but co-ordinated and complementary role for the laminar abscission zone and the petiole during the process of abscission.

In this work, major protein changes in the albedo of the fruit peel of Murcott tangor (tangerine x sweet orange) during postharvest ageing were studied through 2D PAGE. Protein content in matured on-tree fruits and in fruits stored in nonstressing [99% relative humidity (RH) and 25 degrees C], cold (99% RH and 4 degrees C), and drought (60% RH and 25 degrees C) conditions was initially determined. Protein identification through MS/MS determinations revealed in all samples analyzed the occurrence of manganese superoxide dismutase (Mn SOD), actin, ATP synthase beta subunit (ATPase), citrus salt-stress associated protein (CitSap), ascorbate peroxidase (APX), translationally controlled tumor protein (TCTP), and a cysteine proteinase (CP) of the papain family. The latter protein was identified in two different gel spots, with different molecular mass, suggesting the simultaneous presence of the proteinase precursor and its active form. While Mn SOD, actin, ATPase, and CitSap were unchanged in the assayed conditions, TCTP and APX were downregulated during the postharvest ageing process. Ageing-induced APX repression was also reversed by drought. CP contents in albedo, which were similar in on- and off-tree fruits, were strongly dependent upon cold storage. The active/total CP protein ratio significantly increased after cold exposure. This proteomic survey indicates that major changes in protein content in the albedo of the peel of postharvest stored citrus fruits are apparently related to the activation of programmed cell death (PCD).

Carrizo citrange (Citrus sinensisxPoncirus trifoliata) is a citrus hybrid widely used as a rootstock, whose genetic manipulation to improve different growth characteristics is of high agronomic interest. In this work, transgenic Carrizo citrange plants have been produced overexpressing sense and antisense CcGA20ox1 (a key enzyme of GA biosynthesis) under control of the 35S promoter to modify plant architecture. As expected, taller (sense) and shorter (antisense) phenotypes correlated with higher and lower levels, respectively, of active GA1 in growing shoots. In contrast, other phenotypic characteristics seemed to be specific to citrus, or different from those described for similar transgenics in other species. For instance, thorns, typical organs of citrus at juvenile stages, were much longer in sense and shorter in antisense plants, and xylem tissue was reduced in leaf and internode of sense plants. Antisense plants presented a bushy phenotype, suggesting a possible effect of GAs on auxin biosynthesis and/or transport. The main foliole of sense plants was longer, although total leaf area was reduced. Leaf thickness was smaller in sense and larger in antisense plants due to changes in the spongy parenchyma. Internode cell length was not altered in transgenic plants, indicating that, in citrus, GAs regulate cell division rather than cell elongation. Interestingly, the phenotypes described were not apparent when transgenic plants were grafted on non-transgenic rootstock. This suggests that roots contribute to the GA economy of aerial parts in citrus and opens the possibility of using the antisense plants as dwarfing rootstocks.

We analyzed the effects of nitrate availability on growth of Navelina (Citrus sinensis (L.) Osbeck) scions grafted on three citrus rootstocks differing in salt tolerance: Carrizo citrange (Citrus sinensis (L.) Osbeck x Poncirus trifoliata (L.) Raf.), Citrus macrophylla Wester and Cleopatra mandarin (Citrus reshni Hort. ex Tanaka). Salt stress reduced total plant biomass by 27-38%, whereas potassium nitrate supplementation partially counteracted this effect by increasing dry matter and new leaf area. Salinized Carrizo citrange had the greatest response to nitrate supplementation, whereas the effects on salinized Cleopatra mandarin and C. macrophylla were less apparent. Nitrogen and chlorophyll contents and photosynthetic activity also increased in leaves of the nitrate-supplemented salinized plants. In salinized plants, nitrate supplementation reduced leaf abscission, stimulated photosynthetic activity and increased growth of new leaves. The nitrate treatment did not modify chloride concentration in leaves, but it reduced chloride concentrations in Carrizo and Macrophylla roots. Therefore, in both rootstocks, chloride content was similar in mature leaves, higher in immature leaves and lower in roots of the nitrate-supplemented salinized plants compared with salinized plants unsupplemented with nitrate. We suggest that the nitrate-induced stimulation of growth reduced chloride concentration in roots through the reallocation of chloride to new leaves.

We generated source-sink imbalances by defoliation and sucrose supplementation by stem injection, to investigate effects of carbohydrate availability on fruitlet growth and abscission in cv. Okitsu of Satsuma mandarins (Citrus unshiu (Mak.) Marc.). Partial defoliation promoted fruitlet abscission, whereas sucrose supplementation increased citrus fruit set by more than 10%. Moreover, when applied together, sucrose supplementation counteracted the effect of partial defoliation on fruit set. When sucrose was supplied continuously from flowering until harvest, it increased the concentrations of soluble and insoluble sugars in fruits. We conclude that fruit set in citrus is highly dependent on carbohydrate availability.

During ripening, citrus fruit-peel undergoes 'colour break', a process characterized by the conversion of chloroplast to chromoplast. The process involves the progressive loss of chlorophylls and the gain of carotenoids, changing peel colour from green to orange. In the present work, the in vivo and in vitro effects of supplemented nutrients (sucrose and nitrogen) and phytohormones (gibberellins [GA] and ethylene) on colour change in fruit epicarp of Satsuma mandarin (Citrus unshiu (Mak.) Marc., cv. Okitsu), were studied. The rate of colour break was correlated positively with sucrose content and negatively with nitrogen content. The removal of leaves blocked natural sucrose build-up and nitrogen reduction in the peel. Defoliation also inhibited chlorophyll disappearance and carotenoid accumulation, thereby preventing colour break. In vivo sucrose supplementation promoted sucrose accumulation and advanced colour break. In both in vivo and in vitro experiments, colour change promoted by sucrose was unaffected by ethylene but delayed by GA3. In non-supplemented plants, ethylene accelerated colour break while GA3 had no detectable effects. Ethylene inhibitors effectively counteracted the sucrose effects on colour change. Collectively, these results suggest that the chloroplast to chromoplast conversion in citrus fruit epicarps is stimulated by sucrose accumulation. The sugar regulation appears to operate via ethylene, whereas GA may act as a repressor of the sucrose-ethylene stimulation.

The hormonal signals controlling fruitlet abscission induced by sugar shortage in citrus were identified in Satsuma mandarin, Citrus unshiu (Mak.) Marc, cv. Clausellina and cv. Okitsu. Sugar supply, hormonal responses and fruitlet abscission were manipulated through full, partial or selective leaf removals at anthesis and thereafter. In developing fruitlets, defoliations reduced soluble sugars (up to 98%), but did not induce nitrogen and water deficiencies. Defoliation-induced abscission was preceded by rises (up to 20-fold) in the levels of abscisic acid (ABA) and 1-aminocyclopropane-1-carboxylic acid (ACC) in fruitlets. Applications to defoliated plants showed that ABA increased ACC levels (2-fold) and accelerated fruitlet abscission, whereas norflurazon and 2-aminoethoxyvinyl glycine reduced ACC (up to 65%) and fruitlet abscission (up to 40%). Only the full defoliation treatment reduced endogenous gibberellin A1 (4-fold), whereas exogenous gibberellins had no effect on abscission. The data indicate that fruitlet abscission induced by carbon shortage in citrus is regulated by ABA and ACC originating in the fruits, while gibberellins are apparently implicated in the maintenance of growth. In this system, ABA may act as a sensor of the intensity of the nutrient shortage that modulates the levels of ACC and ethylene, the activator of abscission. This proposal identifies ABA and ACC as components of the self-regulatory mechanism that adjusts fruit load to carbon supply, and offers a physiological basis for the photoassimilate competition-induced abscission occurring under natural conditions.

Reproductive and vegetative tissues of the seeded Pineapple cultivars of sweet orange (Citrus sinensis L.) contained the following C-13 hydroxylated gibberellins (GAs): GA53, GA17, GA19, GA20, GA1, GA29, and GA8, as well as GA97, 3-epi-GA1, and several uncharacterized GAs. The inclusion of 3-epi-GA1 as an endogenous substance was based on measurements of the isomerization rates of previously added [2H2]GA1. Pollination enhanced amounts of GA19, GA20, GA29, and GA8 in developing ovaries. Levels of GA1 increased from 5.0 to 9.5 ng/g dry weight during anthesis and were reduced thereafter. The amount of GA in mature pollen was very low. Emasculation reduced GA levels and caused a rapid 100% ovary abscission. This effect was partially counteracted by either pollination or application of GA3. In pollinated ovaries, repeated paclobutrazol applications decreased the amount of GA and increased ovary abscission, although the pattern of continuous decline was different from the sudden abscission induced by emasculation. The above results indicate that, in citrus, pollination increases GA levels and reduces ovary abscission and that the presence of exogenous GA3 in unpollinated ovaries also suppresses abscission. Evidence is also presented that pollination and GAs do not, as is generally assumed, suppress ovary abscission through the reactivation of cell division.

The involvement of abscisic acid (ABA) in the process of leaf abscission induced by 1-aminocyclopropane-1-carboxylic acid (ACC) transported from roots to shoots in Cleopatra mandarin (Citrus reshni Hort. ex Tan.) seedlings grown under water stress was studied using norflurazon (NF). Water stress induced both ABA (24-fold) and ACC (16-fold) accumulation in roots and arrested xylem flow. Leaf bulk ABA also increased (8-fold), although leaf abscission did not occur. Shortly after rehydration, root ABA and ACC returned to their prestress levels, whereas sharp and transitory increases of ACC (17-fold) and ethylene (10-fold) in leaves and high percentages of abscission (up to 47%) were observed. NF suppressed the ABA and ACC accumulation induced by water stress in roots and the sharp increases of ACC and ethylene observed after rewatering in leaves. NF also reduced leaf abscission (7-10%). These results indicate that water stress induces root ABA accumulation and that this is required for the process of leaf abscission to occur. It was also shown that exogenous ABA increases ACC levels in roots but not in leaves. Collectively, the data suggest that ABA, the primary sensitive signal to water stress, modulates the levels of ethylene, which is the hormonal activator of leaf abscission. This assumption implies that root ACC levels are correlated with root ABA amounts in a dependent way, which eventually links water status to an adequate, protective response such as leaf abscission.

Stem elongation and flowering are two processes induced by long-day (LD) treatment in Silene armeria L. Whereas photoperiodic control of stem growth is mediated by gibberellins (GAs), the flowering response cannot be obtained by GA applications. Microscopic observations on early cellular changes in the shoot meristem following LD induction or GA treatment in short days (SD) were combined with GA analyses of stem sections at various distances below the shoot apex. The earliest effects of both LD and GA induction on the subapical meristem were an increase in the number of cells per cell file and a reduction of cell length in the meristematic tissue approx. 1.0-3.0 mm below the shoot apex. Within 8 d after the beginning of LD induction or after GA application, the cells in the subapical meristem were oriented in long files. In induced tips, cellulose deposition occurred mostly in longitudinal walls, indicating that many transverse cell divisions had taken place which, in turn, increased the length of the stem. In contrast to LD induction, GA treatments did not promote the transition from the vegetative to the floral stage. Endogenous GAs were analyzed by selected ion monitoring (SIM), using labeled internal standards, in extracts from transverse sections of the tip at various distances below the apical meristem. In control plants, the levels of the six 13-hydroxy GAs studied (GA53, GA44, GA19, GA20, GA1, and GA8) decreased as the distance from the apical meristem increased. Except for GA53, GA levels were higher in tips of LD-induced plants, particularly in the meristematic zone approx. 0.5-1.5 mm below the apical meristem. In comparison with SD, the highest increase observed was for GA1, the content of which increased 30-fold in the zone 0.5-3.5 mm below the shoot apex. These data indicate a spatial correlation between the accumulation of GA1 and its precursors, and the enhanced mitotic activity which occurs in the subapical meristem of elongating Silene apices.