Improved chilling tolerance in glasshouse-grown potted sweet basil by end-of-production, short-duration supplementary far red light.

Sweet basil is a popular culinary herb used in many cuisines around the world and is widely grown commercially for retail as a live potted plant. However, basil is easily damaged by temperatures below 12 °C meaning plants must be transported from the grower to the retailer in a warm transport chain, adding considerable commercial cost in temperate countries. Improvement of chilling tolerance has been demonstrated in post-harvest crops such as tomato fruits and, indeed, fresh cut basil, by manipulation of the red:far red ratio of light provided to plants throughout the photoperiod and for a significant duration of the growing process in controlled environment chambers. We tested the effectiveness of periodic short-duration end-of-production supplementary far red light treatments designed for use with basil plants grown in a large scale commercial glasshouse for the live potted basil market. Four days of periodic, midday supplementary far red light given at end of production induced robust tolerance to 24 h of 4 °C cold treatment, resulting in greatly reduced visual damage, and reduced physiological markers of chilling injury including electrolyte leakage and reactive oxygen species accumulation. Antioxidant levels were also maintained at higher levels in live potted basil following this cold treatment. RNAseq-based analysis of gene expression changes associated with this response pointed to increased conversion of starch to soluble raffinose family oligosaccharide sugars; increased biosynthesis of anthocyanins and selected amino acids; inactivation of gibberellin signaling; and reduced expression of fatty acid desaturases, all previously associated with increased chilling tolerance in plants. Our findings offer an efficient, non-invasive approach to induce chilling tolerance in potted basil which is suitable for application in a large-scale commercial glasshouse.

Addition of Arbuscular Mycorrhizal Fungi Enhances Terpene Synthase Expression in Salvia rosmarinus Cultivars.

Culinary herbs are commercially cultivated for their wide range of volatile compounds that give characteristic aromas and tastes. Rosemary (Salvia rosmarinus Spenn.) is an excellent model for assessment of methods improvement of volatile production as cultivars offer a wide variety of aromatic profiles due to the large family of terpene synthase genes. Arbuscular mycorrhizal fungi (AMF) associations have been shown to improve essential oil production in aromatic plants and offer one approach to enhance aroma in commercial herb production. Changes in the expression of seven different terpene synthases were compared in six rosemary cultivars in response to addition of AMF to a peat substrate. Addition of AMF profoundly influenced terpene synthase expression in all cultivars and did so without impacting the optimised plant size and uniformity achieved in these conditions. In addition, two methods for AMF application, developed with the horticultural industry in mind, were tested in this study. Uniform incorporation of AMF mixed into the growing substrate prior to planting of a root plug produced the most consistent root colonisation. Overall, our findings demonstrate the potential for the use of AMF in the improvement of aroma in culinary herbs within a commercial setting but show that outcomes are likely to greatly vary depending on variety.

Peduncle Necking in Rosa hybrida Induces Stress-Related Transcription Factors, Upregulates Galactose Metabolism, and Downregulates Phenylpropanoid Biosynthesis Genes.

Roses are highly valued as cut flowers worldwide but have limited vase life. Peduncle bending "bent neck" or "necking" is a major cause of reduced vase life, especially in some cultivars. Necking is thought to be caused by either an air embolism or accumulation of microorganisms at or within the stem end, blocking the xylem vessels and preventing water uptake. However, the underlying mechanisms of necking are poorly understood. Here, RNAseq analysis was applied to compare gene expression across three stages of peduncle necking (straight, <90°, and >90°), in the necking-susceptible Rosa hybrida cultivar H30. Most gene expression change was later in bending and there was, overall, more downregulation than upregulation of gene expression during necking. Photosynthetic, starch, and lignin biosynthesis genes were all downregulated, while genes associated with galactose metabolism, producing raffinose and trehalose that are both related to osmoprotection, were upregulated. Genes associated with starch breakdown, autophagy, and senescence were also upregulated, as were most of the NAC and WRKY transcription factors, involved in stress and senescence regulation. Microscopy showed a cellular collapse in the peduncle. These data support a possible mechanism, whereby a reduction in water transport leads to a cellular collapse in the peduncle, accompanied by upregulation of senescence and drought responses.

Senescence in dahlia flowers is regulated by a complex interplay between flower age and floret position.

Mechanisms regulating flower senescence are not fully understood in any species and are particularly complex in composite flowers. Dahlia (Dahlia pinnata Cav.) florets develop sequentially, hence each composite flower head includes florets of different developmental stages as the whole flower head ages. Moreover, the wide range of available cultivars enables assessment of intraspecific variation. Transcriptomes were compared amongst inner (younger) and outer (older) florets of two flower head ages to assess the effect of floret vs. flower head ageing. More gene expression, including ethylene and cytokinin pathway expression changed between inner and outer florets of older flower heads than between inner florets of younger and older flower heads. Additionally, based on Arabidopsis network analysis, different patterns of co-expressed ethylene response genes were elicited. This suggests that changes occur in young inner florets as the whole flower head ages that are different to ageing florets within a flower head. In some species floral senescence is orchestrated by the plant growth regulator ethylene. However, there is both inter and intra-species variation in its importance. There is a lack of conclusive data regarding ethylene sensitivity in dahlia. Speed of senescence progression, effects of ethylene signalling perturbation, and patterns of ethylene biosynthesis gene expression differed across three dahlia cultivars ('Sylvia', 'Karma Prospero' and 'Onesta') suggesting differences in the role of ethylene in their floral senescence, while effects of exogenous cytokinin were less cultivar-specific.

Fruit shading enhances peel color, carotenes accumulation and chromoplast differentiation in red grapefruit.

The distinctive color of red grapefruits is due to lycopene, an unusual carotene in citrus. It has been observed that red 'Star Ruby' (SR) grapefruits grown inside the tree canopy develop a more intense red coloration than those exposed to higher light intensities. To investigate the effect of light on SR peel pigmentation, fruit were bagged or exposed to normal photoperiodic conditions, and changes in carotenoids, expression of carotenoid biosynthetic genes and plastid ultrastructure in the peel were analyzed. Light avoidance accelerated chlorophyll breakdown and induced carotenoid accumulation, rendering fruits with an intense coloration. Remarkably, lycopene levels in the peel of shaded fruits were 49-fold higher than in light-exposed fruit while concentrations of downstream metabolites were notably reduced, suggesting a bottleneck at the lycopene cyclization in the biosynthetic pathway. Paradoxically, this increment in carotenoids in covered fruit was not mirrored by changes in mRNA levels of carotenogenic genes, which were mostly up-regulated by light. In addition, covered fruits experienced profound changes in chromoplast differentiation, and the relative expression of genes related to chromoplast development was enhanced. Ultrastructural analysis of plastids revealed an acceleration of chloroplasts to chromoplast transition in the peel of covered fruits concomitantly with development of lycopene crystals and plastoglobuli. In this sense, an accelerated differentiation of chromoplasts may provide biosynthetic capacity and a sink for carotenoids without involving major changes in transcript levels of carotenogenic genes. Light signals seem to regulate carotenoid accumulation at the molecular and structural level by influencing both biosynthetic capacity and sink strength.

Underground networking: the potential for improving yield and quality of pot-grown herbs with mycorrhizas.

With constant pressure on herb growers to perform to a continuous high standard, finding new ways to improve herb quality and or quantity are gaining importance, with arbuscular mycorrhizal fungi (AMF) presenting one possible solution. Viviane Schroeder, Alan Gange and Anthony Stead discuss the introduction of AMF to the herb growth cycle and discuss the benefits and costs that their symbiosis with plants bring to modern agriculture.

Proteomic analysis of pollination-induced corolla senescence in petunia.

Senescence represents the last phase of petal development during which macromolecules and organelles are degraded and nutrients are recycled to developing tissues. To understand better the post-transcriptional changes regulating petal senescence, a proteomic approach was used to profile protein changes during the senescence of Petuniaxhybrida 'Mitchell Diploid' corollas. Total soluble proteins were extracted from unpollinated petunia corollas at 0, 24, 48, and 72 h after flower opening and at 24, 48, and 72 h after pollination. Two-dimensional gel electrophoresis (2-DE) was used to identify proteins that were differentially expressed in non-senescing (unpollinated) and senescing (pollinated) corollas, and image analysis was used to determine which proteins were up- or down-regulated by the experimentally determined cut-off of 2.1-fold for P <0.05. One hundred and thirty-three differentially expressed protein spots were selected for sequencing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the identity of these proteins. Searching translated EST databases and the NCBI non-redundant protein database, it was possible to assign a putative identification to greater than 90% of these proteins. Many of the senescence up-regulated proteins were putatively involved in defence and stress responses or macromolecule catabolism. Some proteins, not previously characterized during flower senescence, were identified, including an orthologue of the tomato abscisic acid stress ripening protein 4 (ASR4). Gene expression patterns did not always correlate with protein expression, confirming that both proteomic and genomic approaches will be required to obtain a detailed understanding of the regulation of petal senescence.

A molecular and structural characterization of senescing Arabidopsis siliques and comparison of transcriptional profiles with senescing petals and leaves.

Senescence of plant organs is a genetically controlled process that regulates cell death to facilitate nutrient recovery and recycling, and frequently precedes, or is concomitant with, ripening of reproductive structures. In Arabidopsis thaliana, the seeds are contained within a silique, which is itself a photosynthetic organ in the early stages of development and undergoes a programme of senescence prior to dehiscence. A transcriptional analysis of the silique wall was undertaken to identify changes in gene expression during senescence and to correlate these events with ultrastructural changes. The study revealed that the most highly up-regulated genes in senescing silique wall tissues encoded seed storage proteins, and the significance of this finding is discussed. Global transcription profiles of senescing siliques were compared with those from senescing Arabidopsis leaf or petal tissues using microarray datasets and metabolic pathway analysis software (MapMan). In all three tissues, members of NAC and WRKY transcription factor families were up-regulated, but components of the shikimate and cell-wall biosynthetic pathways were down-regulated during senescence. Expression of genes encoding ethylene biosynthesis and action showed more similarity between senescing siliques and petals than between senescing siliques and leaves. Genes involved in autophagy were highly expressed in the late stages of death of all plant tissues studied, but not always during the preceding remobilization phase of senescence. Analyses showed that, during senescence, silique wall tissues exhibited more transcriptional features in common with petals than with leaves. The shared and distinct regulatory events associated with senescence in the three organs are evaluated and discussed.

A comparison of leaf and petal senescence in wallflower reveals common and distinct patterns of gene expression and physiology.

Petals and leaves share common evolutionary origins but perform very different functions. However, few studies have compared leaf and petal senescence within the same species. Wallflower (Erysimum linifolium), an ornamental species closely related to Arabidopsis (Arabidopsis thaliana), provide a good species in which to study these processes. Physiological parameters were used to define stages of development and senescence in leaves and petals and to align these stages in the two organs. Treatment with silver thiosulfate confirmed that petal senescence in wallflower is ethylene dependent, and treatment with exogenous cytokinin and 6-methyl purine, an inhibitor of cytokinin oxidase, suggests a role for cytokinins in this process. Subtractive libraries were created, enriched for wallflower genes whose expression is up-regulated during leaf or petal senescence, and used to create a microarray, together with 91 senescence-related Arabidopsis probes. Several microarray hybridization classes were observed demonstrating similarities and differences in gene expression profiles of these two organs. Putative functions were ascribed to 170 sequenced DNA fragments from the libraries. Notable similarities between leaf and petal senescence include a large proportion of remobilization-related genes, such as the cysteine protease gene SENESCENCE-ASSOCIATED GENE12 that was up-regulated in both tissues with age. Interesting differences included the up-regulation of chitinase and glutathione S-transferase genes in senescing petals while their expression remained constant or fell with age in leaves. Semiquantitative reverse transcription-polymerase chain reaction of selected genes from the suppression subtractive hybridization libraries revealed more complex patterns of expression compared with the array data.

Hawaiian skirt: an F-box gene that regulates organ fusion and growth in Arabidopsis.

A fast neutron-mutagenized population of Arabidopsis (Arabidopsis thaliana) Columbia-0 wild-type plants was screened for floral phenotypes and a novel mutant, termed hawaiian skirt (hws), was identified that failed to shed its reproductive organs. The mutation is the consequence of a 28 bp deletion that introduces a premature amber termination codon into the open reading frame of a putative F-box protein (At3g61590). The most striking anatomical characteristic of hws plants is seen in flowers where individual sepals are fused along the lower part of their margins. Crossing of the abscission marker, Pro(PGAZAT):beta-glucuronidase, into the mutant reveals that while floral organs are retained it is not the consequence of a failure of abscission zone cells to differentiate. Anatomical analysis indicates that the fusion of sepal margins precludes shedding even though abscission, albeit delayed, does occur. Spatial and temporal characterization, using Pro(HWS):beta-glucuronidase or Pro(HWS):green fluorescent protein fusions, has identified HWS expression to be restricted to the stele and lateral root cap, cotyledonary margins, tip of the stigma, pollen, abscission zones, and developing seeds. Comparative phenotypic analyses performed on the hws mutant, Columbia-0 wild type, and Pro(35S):HWS ectopically expressing lines has revealed that loss of HWS results in greater growth of both aerial and below-ground organs while overexpressing the gene brings about a converse effect. These observations are consistent with HWS playing an important role in regulating plant growth and development.

Ethylene and flower longevity in Alstroemeria: relationship between tepal senescence, abscission and ethylene biosynthesis.

Senescence of floral organs is broadly divided into two groups: those that exhibit sensitivity to exogenous ethylene and those that do not. Endogenous ethylene production from the former group is via a well-characterized biochemical pathway and is either due to developmental or pollination-induced senescence. Many flowers from the order Liliales are characterized as ethylene-insensitive since they do not appear to produce endogenous ethylene, or respond to exogenous ethylene treatments, however, the majority of cases studied are wilting flowers, rather than those where life is terminated by perianth abscission. The role of ethylene in the senescence and abscission of Alstroemeria peruviana cv. Rebecca and cv. Samora tepals was previously unclear, with silver treatments recommended for delaying leaf rather than flower senescence. In the present paper the effects of exogenous ethylene, 2-chloroethylphosphonic acid (CEPA) and silver thiosulphate (STS) treatments on tepal senescence and abscission have been investigated. Results indicate that sensitivity to ethylene develops several days after flower opening such that STS only has a limited ability to delay tepal abscission. Detachment force measurements indicate that cell separation events are initiated after anthesis. Endogenous ethylene production was measured using laser photoacoustics and showed that Alstroemeria senesce independently of ethylene production, but that an extremely small amount of ethylene (0.15 nl flower(-1) h(-1)) is produced immediately prior to abscission. Investigation of the expression of genes involved in ethylene biosysnthesis by semi-quantitative RT-PCR indicated that transcriptional regulation is likely to be at the level of ACC oxidase, and that the timing of ACC oxidase gene expression is coincident with development of sensitivity to exogenous ethylene.

Defining senescence and death.

This article evaluates features of leaf and flower senescence that are shared with, or are different from, those of other terminal events in plant development. Alterations of plastid structure and function in senescence are often reversible and it is argued that such changes represent a process of transdifferentiation or metaplasia rather than deterioration. It may be that the irreversible senescence of many flowers and some leaves represents the loss of ancestral plasticity during evolution. Reversibility serves to distinguish senescence fundamentally from programmed cell death (PCD), as does the fact that viability is essential for the initiation and progress of cell senescence. Senescence (particularly its timing and location) requires new gene transcription, but the syndrome is also subject to significant post- transcriptional and post-translational regulation. The reversibility of senescence must relate to the plastic, facultative nature of underlying molecular controls. Senescence appears to be cell-autonomous, though definitive evidence is required to substantiate this. The vacuole plays at least three key roles in the development of senescing cells: it defends the cell against biotic and abiotic damage, thus preserving viability, it accumulates metabolites with other functions, such as animal attractants, and it terminates senescence by becoming autolytic and facilitating true cell death. The mechanisms of PCD in plants bear a certain relation to those of apoptosis, and some processes, such as nucleic acid degradation, are superficially similar to aspects of the senescence syndrome. It is concluded that, in terms of physiological components and their controls, senescence and PCD are at best only distantly related.

Characterization of a novel lipoxygenase-independent senescence mechanism in Alstroemeria peruviana floral tissue.

The role of lipoxygenase (lox) in senescence of Alstroemeria peruviana flowers was investigated using a combination of in vitro assays and chemical profiling of the lipid oxidation products generated. Phospholipids and galactolipids were extensively degraded during senescence in both sepals and petals and the ratio of saturated/unsaturated fatty acids increased. Lox protein levels and enzymatic activity declined markedly after flower opening. Stereochemical analysis of lox products showed that 13-lox was the major activity present in both floral tissues and high levels of 13-keto fatty acids were also synthesized. Lipid hydroperoxides accumulated in sepals, but not in petals, and sepals also had a higher chlorophyll to carotenoid ratio that favors photooxidation of lipids. Loss of membrane semipermeability was coincident for both tissue types and was chronologically separated from lox activity that had declined by over 80% at the onset of electrolyte leakage. Thus, loss of membrane function was not related to lox activity or accumulation of lipid hydroperoxides per se and differs in these respects from other ethylene-insensitive floral tissues representing a novel pattern of flower senescence.

Cysteine protease gene expression and proteolytic activity during senescence of Alstroemeria petals.

The functional life of the flower is terminated by senescence and/or abscission. Multiple processes contribute to produce the visible signs of petal wilting and inrolling that typify senescence, but one of the most important is that of protein degradation and remobilization. This is mediated in many species through protein ubiquitination and the action of specific protease enzymes. This paper reports the changes in protein and protease activity during development and senescence of Alstroemeria flowers, a Liliaceous species that shows very little sensitivity to ethylene during senescence and which shows perianth abscission 8-10 d after flower opening. Partial cDNAs of ubiquitin (ALSUQ1) and a putative cysteine protease (ALSCYP1) were cloned from Alstroemeria using degenerate PCR primers and the expression pattern of these genes was determined semi-quantitatively by RT-PCR. While the levels of ALSUQ1 only fluctuated slightly during floral development and senescence, there was a dramatic increase in the expression of ALSCYP1 indicating that this gene may encode an important enzyme for the proteolytic process in this species. Three papain class cysteine protease enzymes showing different patterns of activity during flower development were identified on zymograms, one of which showed a similar expression pattern to the cysteine protease cDNA.