Artificial Pollination in Kiwifruit and Olive Trees

In the last 10 years, kiwifruit vine artificial pollination became a widespread practice useful to increase fruit quality. Kiwifruit size is directly proportional to the number of seeds, i.e., to the number of fertilized ovaries. However, artificial pollination efficiency depends on many parameters such as pollen quality (germinability, humidity, and conservation), pollination system (dry or liquid), coadjuvants, and flowering stage. Those parameters were well defined in Actinidia in recent studies, however, they remain quite undefined for other anemophilous pollinated trees such as olive tree, hazelnut, pistachio, and palm. In these plants, the flowers are very small and extremely numerous, so the pollination was difficult to study. In addition, there are incompatibility factors (genetic and physic), long lap time from pollination to fertilization, and alternate bearing, lower economic gain for these fruits, low agronomic input, and low innovation level in the field. All these aspects had reduced the application of pollination technique for these cultivations. The experiences developed in kiwifruit lead to define a new model crop fruit set that could be applied to anemophilous pollinated plants such as olive tree, where the fruit set are lower than 2%. The first experiences have shown a great potential and have encouraged the development of this technique

Comparative transcriptome analysis of the interaction between Actinidia chinensis var. chinensis and Pseudomonas syringae pv. Actinidiae in absence and presence of acibenzolar-S-methyl

Background: Since 2007, bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) has become a pandemic disease leading to important economic losses in every country where kiwifruit is widely cultivated. Options for controlling this disease are very limited and rely primarily on the use of bactericidal compounds, such as copper, and resistance inducers. Among the latter, the most widely studied is acibenzolar-S-methyl. To elucidate the early molecular reaction of kiwifruit plants (Actinidia chinensis var. chinensis) to Psa infection and acibenzolar-S-methyl treatment, a RNA seq analysis was performed at different phases of the infection process, from the epiphytic phase to the endophytic invasion on acibenzolar-S-methyl treated and on non-treated plants. The infection process was monitored in vivo by confocal laser scanning microscopy. Results: De novo assembly of kiwifruit transcriptome revealed a total of 39,607 transcripts, of which 3360 were differentially expressed during the infection process, primarily 3 h post inoculation. The study revealed the coordinated changes of important gene functional categories such as signaling, hormonal balance and transcriptional regulation. Among the transcription factor families, AP2/ERF, MYB, Myc, bHLH, GATA, NAC, WRKY and GRAS were found differentially expressed in response to Psa infection and acibenzolar-S-methyl treatment. Finally, in plants treated with acibenzolar-S-methyl, a number of gene functions related to plant resistance, such as PR proteins, were modulated, suggesting the set-up of a more effective defense response against the pathogen. Weighted-gene coexpression network analysis confirmed these results. Conclusions: Our work provides an in-depth description of the plant molecular reactions to Psa, it highlights the metabolic pathway related to acibenzolar-S-methyl-induced resistance and it contributes to the development of effective control strategies in open field

Plant Microbiome and Its Link to Plant Health: Host Species, Organs and Pseudomonas syringae pv. actinidiae Infection Shaping Bacterial Phyllosphere Communities of Kiwifruit Plants

Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of the bacterial canker, the most devastating disease of kiwifruit vines. Before entering the host tissues, this pathogen has an epiphytic growth phase on kiwifruit flowers and leaves, thus the ecological interactions within epiphytic bacterial community may greatly influence the onset of the infection process. The bacterial community associated to the two most important cultivated kiwifruit species, Actinidia chinensis and Actinidia deliciosa, was described both on flowers and leaves using Illumina massive parallel sequencing of the V3 and V4 variable regions of the 16S rRNA gene. In addition, the effect of plant infection by Psa on the epiphytic bacterial community structure and biodiversity was investigated. Psa infection affected the phyllosphere microbiome structures in both species, however, its impact was more pronounced on A. deliciosa leaves, where a drastic drop in microbial biodiversity was observed. Furthermore, we also showed that Psa was always present in syndemic association with Pseudomonas syringae pv. syringae and Pseudomonas viridiflava, two other kiwifruit pathogens, suggesting the establishment of a pathogenic consortium leading to a higher pathogenesis capacity. Finally, the analyses of the dynamics of bacterial populations provided useful information for the screening and selection of potential biocontrol agents against Psa

Physical Mapping of Bread Wheat Chromosome 5A: An Integrated Approach

The huge size, redundancy, and highly repetitive nature of the bread wheat [Triticum aestivum (L.)] genome, makes it among the most difficult species to be sequenced. To overcome these limitations, a strategy based on the separation of individual chromosomes or chromosome arms and the subsequent production of physical maps was established within the frame of the International Wheat Genome Sequence Consortium (IWGSC). A total of 95,812 bacterial artificial chromosome (BAC) clones of short-arm chromosome 5A (5AS) and long-arm chromosome 5A (5AL) arm-specific BAC libraries were fingerprinted and assembled into contigs by complementary analytical approaches based on the FingerPrinted Contig (FPC) and Linear Topological Contig (LTC) tools. Combined anchoring approaches based on polymerase chain reaction (PCR) marker screening, microarray, and sequence homology searches applied to several genomic tools (i. e., genetic maps, deletion bin map, neighbor maps, BAC end sequences (BESs), genome zipper, and chromosome survey sequences) allowed the development of a high-quality physical map with an anchored physical coverage of 75% for 5AS and 53% for 5AL with high portions (64 and 48%, respectively) of contigs ordered along the chromosome. In the genome of grasses, Brachypodium [Brachypodium distachyon (L.) Beauv.], rice (Oryza sativa L.), and sorghum [Sorghum bicolor (L.) Moench] homologs of genes on wheat chromosome 5A were separated into syntenic blocks on different chromosomes as a result of translocations and inversions during evolution. The physical map presented represents an essential resource for fine genetic mapping and map-based cloning of agronomically relevant traits and a reference for the 5A sequencing projects

Physiological and molecular basis of meristematic and embryogenic competence displayed by the epiphyllous clone EMB-2 of Helianthus

In higher plants, the zygote divides to produce the embryo, a bipolar structure with one, two or several embryonic leaves (cotyledons), the shoot apical meristem (SAM) and the root apical meristem (RAM). Throughout plant life, apical meristems continually produce new organs (branches, leaves or roots), which are continuously added to the plant body. An important consequence of this unlimited and reiterative growth, defined as “recurrent ontogenesis”, is the absence of separation between germ-line and soma; thus any somatic cell is a potential progenitor of a new individual. This capacity (totipotency) is revealed by processes of vegetative propagation in vivo, such as: i) adventitious embryos from nucellar cells; ii) adventitious embryos from single cells of epidermis of stems and leaves; iii) adventitious buds of multicellular origin. Moreover, somatic cell totipotency is most clearly expressed in the ability of plant cells to develop into a complete fertile plants by in vitro adventitious embryogenesis and/or organogenesis. The concept of totipotency implies that every plant cell, with a normal complement of chromosomes, is able to express its genetic potential by regenerating a whole plant. However, as differentiation proceeds, tissues, initially competent to develop in a number of different patterns, reduce their degree of freedom, becoming eventually determined with respect to a single fate. Differentiated plant cells can also acquire new cell fates in the adult body, often independently of neighbouring cells. Structures as embryos, shoots, leaves and flowers may occur upon a leaf or leaf homologue in any position (epiphylly). The principal aim of this work is the improvement of our information on plant cell totipotency at molecular, cytological and biochemical level through the study of a plant systems featured by epiphylly: the clone EMB-2 of the interspecific hybrid Helianthus annuus x H. tuberosus characterized by the ectopic proliferation of adventitious morphogenetic structures on leaves. Notwithstanding the relative difficulty to identify the genes involved in the acquisition of cell totipotency, it is likely that a regulatory network, analogous to that involved in meristem and zygotic embryo development control the coordinate development of adventitious meristems and somatic embryos. Recent findings seem to support this point of view. In fact, the ectopic expression of genes involved in formation and maintenance of SAM (e.g. KNOTTED1-like homeobox, KNOX) or in embryo development (e.g. LEAFY COTYLEDON, LEC) is sufficient to re-programme in vivo the fate of differentiate cells. In the present work, the LEAFY COTYLEDON1-like gene of H. annuus (HaL1L) was isolated HaL1L expression patterns. HaL1L mRNA peaked at an early stage of embryogenesis (i.e. globular-, heart-, early cotyledon-embryo). More precisely, at the globular and heart stage HaL1L mRNA was prevalent in the outer cell layers of the embryo, in the suspensor and in integumentary tapetum cells. At cotyledon stage, HaL1L transcripts became evenly distributed throughout the embryo, but the positive signal was more evident in both inner cells of the integument and integumentary tapetum cells. In particular, a very strong signal was detected in the chalaza’s window. This result could support the hypothesis that HaL1L plays a role in the control of the nutrient transfer during embryo development. The class I KNOX gene of H. tuberosus, named HtKNOT1, was also isolated. HtKNOT1 was expressed in vegetative shoot apices and stem internodes of H. tuberosus, while leaves (blades and veins) and petioles did not accumulate any detectable HtKNOT1 transcripts. In particular, a higher accumulation of HtKNOT1 mRNA was detected in apical stem internodes in comparison to medial and basal region of the stem. HtKNOT1 transcripts were strongly detected in the meristematic dome of the SAM, but a weak presence of transcripts was also detected in incipient leaf primordia. These findings indicate that, in some species, the transcriptional down-regulation of class I KNOX genes is not a prerequisite for lateral organ initiation. In apical stem internodes of H. tuberosus, HtKNOT1 is expressed in cambial cells, phloem cells and xylematic parenchyma cells of the xylem. In basal internodes, HtKNOT1 expression was restricted to the presumptive initials and recently derived phloem cells. The expression of HtKNOT1 in the cambium is not surprising because the class I KNOX genes are involved in the meristem maintenance and the cambial cells behave as totipotent cells responsible for the stem radial growth. In this work we have also investigated the possible role of HtKNOT1 in controlling the development of the heterogamous inflorescence in the Helianthus genus. Inflorescence meristems of H. annuus and H. tuberosus showed HtKNOT1 expression in the region of the floret meristems and in developing organ primordia (i.e. floral bracts, petals, stamens and carpels). In older flowers strong expression of HtKNOT1 was seen in developing ovule. Notably, in the anthers of H. annuus, HtKNOT1 expression was also seen in pollen mother cells, in the tapetum and in the first and second mitotic division of developing pollen. This expression analysis also strongly suggests that the HtKNOT1 function is employed in pollen morphogenesis: this fact, if confirmed, will be one not yet discovered role of the class I KNOX genes. Several data support the view that when class I KNOX and/or LEC genes are not properly repressed adventitious phenomena of epiphylly became possible in different species. The EMB-2 somaclonal variant, derived by in vitro tissue culture of the tetraploid (2n = 4x = 68) interspecific hybrid Helianthus annuus x H. tuberosus (A-2 clone), shows an unusual pattern of development: it produces both in vitro and in vivo, epiphyllous embryos and shoot-like structures. More precisely, in addition to nonepiphyllous leaves (NEP) that expanded normally, some leaves of EMB-2 plants exhibit on the adaxial surface knobs and a prominent proliferation of ectopic structures (EP leaves), usually arranged in clusters along leaf veins. In EMB-2 plants, HtKNOT1 was highly expressed in both stem internodes and EP leaves. By contrast, HtKNOT1 mRNA accumulation was undetected in roots, petioles and NEP leaves. HtKNOT1 transcripts were confined at the level of the palisade cell layer in EP leaves. Thereafter, transcripts accumulated in developing morphogenetic structures, but the signal did not spread to epidermal cells. However, as development of shoot-like structures proceeded, transcript signal was also spread throughout epidermal cells. Afterwards, the level of Ht-KNOT1 transcripts decreased and mainly accumulated in the external cell layers of ectopic shoot-like structures. In contrast, in adventitious embryos, HtKNOT1 transcripts were confined to the basal portion of structures, whereas in the advanced globular embryos, the signal was restricted to a few scattered cellsHaL1L transcripts were undetected in both A-2 and NEP leaves while it was expressed in EP leaves. More precisely, the accumulation of HaL1L transcripts was evenly detected throughout all stages of somatic embryos. On the contrary, no signal related to HaL1L transcript accumulation was observed in developing shoot-like structures. Histological analyses suggested that epidermal and parenchyma cells around the vascular bundles contributed to the development of epiphyllous structures. Thus, the ectopic accumulation of HaL1L mRNA in this cell type opens the possibility that HaL1L is also involved in switching somatic cell fate towards embryogenic competence. Given the clear link of both cytokinins and auxin with plant organogenesis and embryogenesis, the endogenous levels of these hormones were detected in epiphyllous (EP) and non-epiphyllous (NEP) leaves of EMB-2 plants as well in the non-epiphyllous control genotype (A-2). The endogenous levels of cytokinins and IAA in NEP leaves of EMB-2 plants did not significantly differ in comparison to A-2 leaves. By contrast, the content of both hormones in EP leaves were 1.8-fold higher than NEP ones. The results suggested that epiphylly of EMB-2 plants was tightly related to a localized increase of hormones in EP leaves as compared to NEP ones. The involvement of cytokinins in ectopic morphogenetic events of EMB-2 plants was strengthened by immunolocalization results. The presence of active zeatin marked the development of adventitious shoot-like structures, from the first division of epidermal cells to SAM establishment. In EP leaves, a strong immunostaining was detected wherever the ectopic structures were formed, as well as at the level of the vascular bundles. In addition, a strong immunoreaction marked adaxial epidermal cells, which in some case undergo periclinal division, as well as clusters of epidermal cells, from which morphogenetic structures were likely to originate. As morphogenetic structures developed, a strong immunostaining was clearly detected. By contrast, in epiphyllous embryolike structures, zeatin signals were confined to a few scattered cells. Notably, before ectopic structures became evident, HtKNOT1 expression and zeatin signal were localized in distinct histological domains (mesophyll tissue vs. epidermis). However, as morphogenetic processes occurred, the accumulation of HtKNOT1 transcripts and zeatin clearly overlapped. A-2 leaves (i.e. non epiphyllous clone) displayed a strong accumulation of auxin in vascular bundles but not in the epidermis. Nevertheless, an immunostaining signal of indole-3-acetic acid (IAA) was evident in the mesophyll cells, preferentially in the chloroplasts. By contrast, EP leaves showed an auxin accumulation in a single cell or in a small group of cells of the adaxial epidermis. Indeed, early stages of ectopic embryogenic structures showed a strong immunostaining at the level of the whole globular embryo-like structure. By contrast, as for the zeatin immunolocalization, in advanced stages of embryo development a characteristic scattered immunoreaction was observed. In EP leaves with shoot-like structures, the auxin is preferentially accumulated in the protoderm and in active dividing cells. Interestingly, in the early stages of somatic embryo development, HaL1L expression and auxin were localized in the same histological domains (i.e. epidermis, parenchyma cells of vascular bundles, globular embryos). In conclusion, the epiphylly of EMB-2 plants seems linked to a complex regulatory network involving hormones and transcription factors. At physiological level the epiphylly of EMB-2 can be related to a localized increase of both zeatin and IAA while about the molecular mechanisms involved, HtKNOT1 and HaL1L cooperate during ectopic embryogenesis. On the contrary, in adventitious organogenesis only misexpression of HtKNOT1 has probably specific function. The histological analyses, the localization of auxin and cytokinin in specific histological domains and the expression patterns of HaL1L and HtKNOT1 also identified the epidermal cells located in proximity of vascular bundles as the totipotent cells of the EMB-2 clone. The localized increase of IAA in epidermal layers of EP leaves could determines an alteration in the intracellular polarity an therefore a shift of cell division plane (anticlinal vs. periclinal). Thereafter, citokinins, activated by HtKNOT1, could promote, by active divisions, the development of ectopic structures. Additionally, the two transcription factors could interact with other regulators of embryogenesis and/or organogenesis in the differentiation and organization of ectopic structure

stem fasciated, a Recessive Mutation in Sunflower (Helianthus annuus), Alters Plant Morphology and Auxin Level

• Background and Aims Plant lateral organs such as leaves arise from a group of initial cells within the flanks of the shoot apical meristem (SAM). Alterations in the initiation of lateral organs are often associated with changes in the dimension and arrangement of the SAM as well as with abnormal hormonal homeostasis. A mutation named stem fasciated (stf) that affects various aspects of plant development, including SAM shape and auxin level, was characterized in sunflower (Helianthus annuus)

Regulation and Evolution of NLR Genes: A Close Interconnection for Plant Immunity

NLR (NOD-like receptor) genes belong to one of the largest gene families in plants. Their role in plants’ resistance to pathogens has been clearly described for many members of this gene family, and dysregulation or overexpression of some of these genes has been shown to induce an autoimmunity state that strongly affects plant growth and yield. For this reason, these genes have to be tightly regulated in their expression and activity, and several regulatory mechanisms are described here that tune their gene expression and protein levels. This gene family is subjected to rapid evolution, and to maintain diversity at NLRs, a plethora of genetic mechanisms have been identified as sources of variation. Interestingly, regulation of gene expression and evolution of this gene family are two strictly interconnected aspects. Indeed, some examples have been reported in which mechanisms of gene expression regulation have roles in promotion of the evolution of this gene family. Moreover, co-evolution of the NLR gene family and other gene families devoted to their control has been recently demonstrated, as in the case of miRNAs

Comparative transcriptome analysis of the interaction between Actinidia chinensis var. chinensis and Pseudomonas syringae pv. actinidiae in absence and presence of acibenzolar-S-methyl

Abstract Background Since 2007, bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) has become a pandemic disease leading to important economic losses in every country where kiwifruit is widely cultivated. Options for controlling this disease are very limited and rely primarily on the use of bactericidal compounds, such as copper, and resistance inducers. Among the latter, the most widely studied is acibenzolar-S-methyl. To elucidate the early molecular reaction of kiwifruit plants (Actinidia chinensis var. chinensis) to Psa infection and acibenzolar-S-methyl treatment, a RNA seq analysis was performed at different phases of the infection process, from the epiphytic phase to the endophytic invasion on acibenzolar-S-methyl treated and on non-treated plants. The infection process was monitored in vivo by confocal laser scanning microscopy. Results De novo assembly of kiwifruit transcriptome revealed a total of 39,607 transcripts, of which 3360 were differentially expressed during the infection process, primarily 3 h post inoculation. The study revealed the coordinated changes of important gene functional categories such as signaling, hormonal balance and transcriptional regulation. Among the transcription factor families, AP2/ERF, MYB, Myc, bHLH, GATA, NAC, WRKY and GRAS were found differentially expressed in response to Psa infection and acibenzolar-S-methyl treatment. Finally, in plants treated with acibenzolar-S-methyl, a number of gene functions related to plant resistance, such as PR proteins, were modulated, suggesting the set-up of a more effective defense response against the pathogen. Weighted-gene coexpression network analysis confirmed these results. Conclusions Our work provides an in-depth description of the plant molecular reactions to Psa, it highlights the metabolic pathway related to acibenzolar-S-methyl-induced resistance and it contributes to the development of effective control strategies in open field