Current status and future challenges in implementing and upscaling vertical farming systems.

Vertical farming can produce food in a climate-resilient manner, potentially emitting zero pesticides and fertilizers, and with lower land and water use than conventional agriculture. Vertical farming systems (VFS) can meet daily consumer demands for nutritious fresh products, forming a part of resilient food systems-particularly in and around densely populated areas. VFS currently produce a limited range of crops including fruits, vegetables and herbs, but successful implementation of vertical farming as part of mainstream agriculture will require improvements in profitability, energy efficiency, public policy and consumer acceptance. Here we discuss VFS as multi-layer indoor crop cultivation systems, exploring state-of-the-art vertical farming and future challenges in the fields of plant growth, product quality, automation, robotics, system control and environmental sustainability and how research and development, socio-economic and policy-related institutions must work together to ensure successful upscaling of VFS to future food systems.

Involvement of ethylene and lipid signalling in cadmium-induced programmed cell death in tomato suspension cells.

Cadmium-induced cell death was studied in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells (line MsK8) treated with CdSO(4). Within 24 h, cadmium treatment induced cell death in a concentration-dependent manner. Cell cultures showed recovery after 2-3 days which indicates the existence of an adaptation mechanism. Cadmium-induced cell death was alleviated by the addition of sub muM concentrations of peptide inhibitors specific to human caspases indicating that cell death proceeds through a mechanism with similarities to animal programmed cell death (PCD, apoptosis). Cadmium-induced cell death was accompanied by an increased production of hydrogen peroxide (H(2)O(2)) and simultaneous addition of antioxidants greatly reduced cell death. Inhibitors of phospholipase C (PLC) and phospholipase D (PLD) signalling pathway intermediates reduced cadmium-induced cell death. Treatment with the G-protein activator mastoparan and a cell permeable analogue of the lipid signal second messenger phosphatidic acid (PA) induced cell death. Ethylene, while not inducing cell death when applied alone, stimulated cadmium-induced cell death. Application of the ethylene biosynthesis inhibitor aminoethoxy vinylglycine (AVG) reduced cadmium-induced cell death, and this effect was alleviated by simultaneous treatment with ethylene. Together the results show that cadmium induces PCD exhibiting apoptotic-like features. The cell death process requires increased H(2)O(2) production and activation of PLC, PLD and ethylene signalling pathways.

Controlling ethylene responses in flowers at the receptor level.

For a vast number of ornamental species, blocking the plant's response to ethylene is an efficient strategy to enhance the longevity of the flowers. The most effective ways to conduct such interference will be reviewed in this paper. A large number of chemical compounds have been evaluated for their effects on ethylene production and perception. Among these are a range of strained olefines. This has resulted in the discovery that cyclopropenes, among them 1-methylcyclopropene (1-MCP) and a number of other substituted cyclopropenes effectively block ethylene responses at the receptor level. A lot of testing remains to be done to uncover the full potential of these compounds, but they do offer promising new ways to extend the postharvest life of ornamentals. Also genetic modification appears to be a very effective way in controlling of ethylene synthesis and perception. Attempts to use both a reduced endogenous ethylene production and a reduced sensitivity to ethylene will be reviewed. Among these the use of the mutant ethylene receptor gene, etr1-1, from Arabidopsis seems most promising, especially when it is expressed under the control of a flower specific promoter.

Gravitropism in cut flower stalks of snapdragon.

The negative gravitropic response of cut flower stalks is a complex multistep process that requires the participation of various cellular components acting in succession or in parallel. The process was particularly characterized in snapdragon (Antirrhinum majus L.) spikes with regard to (1) gravity stimulus perception associated with amyloplast reorientation; (2) stimulus transduction mediated through differential changes in the level, action and related genes of auxin and ethylene and their possible interaction; (3) stimulus response associated with differential growth leading to stalk curvature; (4) involvement of cytosolic calcium and actin cytoskeleton. Results show that the gravity-induced amyloplast reorientation, differential over-expression of two early auxin responsive genes and asymmetrical distribution of free IAA are early events in the bending process. These precede the asymmetrical ethylene production and differential stem growth, which was derived from initial shrinkage of the upper stem side and a subsequent elongation of the lower stem side. Results obtained with various calcium- and cytoskeleton-related agents indicate that cytosolic calcium and actin filaments may play essential roles in gravitropism-related processes of cut flower stalks. Therefore, modulators of these two physiological mediators may serve as means for controlling any undesired gravitropic bending.

A tomato homologue of the human protein PIRIN is induced during programmed cell death.

Programmed cell death (PCD), with similarities to animal apoptosis, was induced in tomato suspension cells by the topoisomerase I inhibitor camptothecin. Previously, a differential display screening was performed to isolate genes differentially expressed during camptothecin-induced cell death. As a result, the new tomato gene Le-pirin was isolated, whose mRNA levels dramatically increase during camptothecin-induced PCD. Le-pirin mRNA accumulation is also observed when cell death is triggered by the mycotoxin fumonisin-B1, but not when the suspension cells are treated with stress-related compounds such as ethylene, methyl jasmonate or salicylic acid. The caspase inhibitor Z-Asp-CH2-DCB and the calcium channel blocker LaCl3 effectively delayed whereas ethylene greatly stimulated camptothecin-induced PCD and the accumulation of Le-pirin mRNA. The Le-pirin encoded protein shows 56% identity with the human protein PIRIN, a nuclear factor reported to interact with the human oncogene Bcl-3. Human PIRIN stabilizes the formation of quaternary complexes between Bcl-3, the anti-apoptotic transcription factor NF-kappaB and its DNA target sequences in vitro. The isolation of Le-pirin and its implication in plant PCD provides new clues on the role of putative NF-kappaB-associated pathways in plant defence mechanisms.

Changes in gene expression during programmed cell death in tomato cell suspensions.

To identify genes involved in plant programmed cell death (PCD), changes in gene expression during PCD in a model system of suspension-cultured tomato cells were studied. In this system, cell death is triggered by treatment with camptothecin, an inhibitor of topoisomerase 1. Cell death was accompanied by internucleosomal DNA degradation, indicating that the cell death process shares similarities with apoptosis in animals. Tomato homologues of DAD1 and HSR203, two genes that have been implicated in PCD, were isolated. During camptothecin-induced PCD tomato DAD1 mRNA levels roughly halve, while tomato HSR203 mRNA levels increase 5-fold. A differential display approach was used to identify novel genes that show changes in expression levels during camptothecin-induced PCD. This resulted in isolation of two up-regulated (CTU1 and CTU2) and four down-regulated (CTD1, CTD2, CTD4, and CTD5) cDNA clones. CTU1 shows high homology to various gluthatione S-transferases, whereas CTU2 is as yet unidentified. CTD1 is highly similar to Aux/IAA early-auxin-responsive genes. CTD2 corresponds to the tomato RSI-I gene, CTD4 is an unknown clone, and CTD5 shows limited homology with a proline-rich protein from maize. Addition of the calcium channel blocker lanthanum chloride prevented camptothecin-induced cell death. The effect of lanthanum chloride on camptothecin-induced gene expression was studied to discriminate between putative cell death genes and general stress genes. The possible role of the various predicted gene products in plant PCD is discussed.

Chemical-induced apoptotic cell death in tomato cells: involvement of caspase-like proteases.

A new system to study programmed cell death in plants is described. Tomato (Lycopersicon esculentum Mill.) suspension cells were induced to undergo programmed cell death by treatment with known inducers of apoptosis in mammalian cells. This chemical-induced cell death was accompanied by the characteristic features of apoptosis in animal cells, such as typical changes in nuclear morphology, the fragmentation of the nucleus and DNA fragmentation. In search of processes involved in plant apoptotic cell death, specific enzyme inhibitors were tested for cell-death-inhibiting activity. Our results showed that proteolysis plays a crucial role in apoptosis in plants. Furthermore, caspase-specific peptide inhibitors were found to be potent inhibitors of the chemical-induced cell death in tomato cells, indicating that, as in animal systems, caspase-like proteases are involved in the apoptotic cell death pathway in plants.

Ethylene biosynthetic genes are differentially expressed during carnation (Dianthus caryophyllus L.) flower senescence.

Ethylene production and expression patterns of an 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (CARAO1) and of two ACC synthase (EC 4.4.1.14) genes (CARACC3 and CARAS1) were studied in floral organs of cut carnation flowers (Dianthus caryophyllus L.) cv. White Sim. During the vase life and after treatment of fresh flowers with ethylene, production of ethylene and expression of ethylene biosynthetic genes first started in the ovary followed by the styles and the petals. ACC oxidase was expressed in all the floral organs whereas, during the vase life, tissue-specific expression of the two ACC synthase genes was observed. After treatment with a high ethylene concentration, tissue specificity of the two ACC synthase genes was lost and only a temporal difference in expression remained. In styles, poor correlation between ethylene production and ACC synthase (CARAS1) gene expression was observed suggesting that either activity is regulated at the translational level or that the CARAS1 gene product requires an additional factor for activity. Isolated petals showed no increase in ethylene production and expression of ethylene biosynthetic genes when excised from the flower before the increase in petal ethylene production (before day 7); showed rapid cessation of ethylene production and gene expression when excised during the early phase of petal ethylene production (day 7) and showed a pattern of ethylene production and gene expression similar to the pattern observed in the attached petals when isolated at day 8. The interorgan regulation of gene expression and ethylene as a signal molecule in flower senescence are discussed.

The Role of Ethylene in Interorgan Signaling during Flower Senescence.

The roles of 1-aminocyclopropane-1-carboxylic acid (ACC) and ethylene in interorgan signaling during senescence in orchid (Cymbidium) flowers were investigated. Following application of radiolabeled ACC to the stigma or the rostellum (modified lobe of the stigma), radiolabeled ethylene is produced by all flower parts. In intact flowers as well as in excised central columns, stigma- or rostellum-applied ACC or [alpha]-aminoisobutyric acid were largely immobile. Local treatment of the central column of previously aminoethoxyvinylglycine-treated flowers with either ethylene or 2-chloroethylphosphonic acid (ethephon) rapidly induced emission of ethylene from the petals, showing that ethylene is readily translocated within the flower. Creation of alternative outlets (incisions) in the labellum or the central column significantly delayed the occurrence of senescence symptoms in ACC-treated flowers. The results do not confirm the presumed role of ACC as a signal in interorgan communication during flower senescence. In these flowers, ethylene produced in the stigmatic region following pollination or emasculation serves as a mobile factor responsible for senescence symptoms observed in other flower parts.

Investigation of Local Ethylene Emission from Intact Cherry Tomatoes by Means of Photothermal Deflection and Photoacoustic Detection.

The function of the coronet region of the cherry tomato (Lycopersicon esculentum Cherry) as the main emission channel for ethylene was investigated. Ethylene was measured employing two laser-based detection systems, the photothermal deflection instrument and the photoacoustic detection setup. It is possible to detect minimum ethylene concentrations of 1 nL L-1 locally and rapidly with the first instrument and concentrations of 6 pL L-1 in a continuous flow system with the second setup. The continuous flow system makes it possible to change the air composition and to monitor its influence on the ethylene production of the tomato. The response times of the two instruments are 30 s and 4 min, respectively. The local character of the measurements makes it possible to determine the emission sites of the gaseous plant hormone ethylene and their relative importance. Transient anoxic conditions stop production of ethylene; return to aerobic conditions shows the evolution of the accumulated ethylene precursor 1-aminocyclopropane-1-carboxylic acid and its renewed production on the measured ethylene emission, with a time resolution of several minutes. Temporarily sealing the main emission channel yields results comparable to anoxia.

Reactions of cloned poplars to air pollution : Ozone-induced increase of stress ethylene and possible antisenescence strategies.

Ozone-induced changes in ethylene production, ACC oxidase activity and the contents of ACC, MACC and free PAs were studied inPopulus nigra L. cv. Loenen with high ozone sensitivity as judged by the degree of chlorophyll degradation and premature leaf abscission.Ethylene production, ACC oxidase activity, ACC content and MACC levels were induced by the one-, two-, and three-week ozone exposure (36±9 ppb O3 for 11 hours a day). In addition, increases in PA levels, especially in spermidine, were measured in ozone treated plants.The role of free PAs and MACC synthesis as possible antisenescence reactions is discussed.

Molecular cloning of two different ACC synthase PCR fragments in carnation flowers and organ-specific expression of the corresponding genes.

Degenerate oligonucleotides to highly conserved regions of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14), the key enzyme in ethylene biosynthesis, were used to prime the synthesis and amplification of fragments of about 1,180 bp by polymerase chain reaction (PCR) in samples of cDNA to total RNA isolated from senescing carnation (Dianthus caryophyllus) flowers. Two putative ACC synthase PCR clones were isolated one of which was identical to the sequence of a carnation ACC synthase cDNA clone (CARACC3) recently isolated by Park et al. (Plant Mol Biol 18 (1992) 377-386). The other clone (CARAS1) was ca. 66% homologous at the amino acid level to CARACC3. For both ACC synthase clones, specific oligonucleotides were synthesized and, using PCR, we were able to distinguish between the two ACC synthase transcripts in samples of total RNA isolated from different carnation flower parts and leaves. DNA blots of PCR fragments revealed that, in flowers, both ageing and ethylene stimulated the occurrence of these transcripts in an organ-specific way. CARACC3 was more abundant in RNA from the petals whereas CARAS1 was more abundant in RNA from the styles. Despite a high ethylene production observed in ovaries, the level of both transcripts was low, suggesting the existence of a third ACC synthase gene that is specifically expressed in the ovary. Transcript levels in leaves were low irrespective of treatment.

Behavior of Etiolated Peas (Pisum sativum cv Alaska) When Obstructed by a Mechanical Barrier.

Etiolated pea (Pisum sativum cv Alaska) seedlings growing against a horizontal barrier in the soil will assume a horizontal orientation and continue to grow for prolonged periods of time. With removal of the barrier or after seedlings grow out from underneath the obstruction, seedlings immediately return to normal vertical growth. Ethylene production increased several hours after the seedlings began to grow horizontally and not at the first contact with a barrier. Increases in ethylene production from horizontally growing seedlings were associated with decreased rates of elongation and increased stem diameter. The data suggest that increased ethylene production does not play a mediating role in the horizontal growth of pea seedlings when obstructed during emergence. We conclude that seedlings follow a path of least resistance when they grow against a barrier in the soil.

Interorgan translocation of 1-aminocyclopropane-1-carboxylic Acid and ethylene coordinates senescence in emasculated cymbidium flowers.

In Cymbidium flowers, emasculation by removal of the pollinia and the anther cap leads within 24 hours to red coloration of the labellum (lip). Lip coloration, being the first sign of senescence in these flowers, has been ascribed to the action of ethylene in the lip. When a small incision in the base of the lip is made prior to emasculation, or when the lip is excised and placed in water within 10 to 15 hours after emasculation, coloration is considerably delayed. This indicates that a coloration-associated factor is moving in or out of the lip. Measurements of ethylene production of excised flower parts, isolated at different times after emasculation, showed an increase only in the central column; the other flower parts, including the lip, did not show a measurable change. In contrast, in situ measurements of the ethylene production of the central column and the remaining portion of the flower revealed a simultaneous increase in all the flower parts following emasculation. Similarly, application of radiolabeled 1-aminocyclopropane-1-carboxylic acid (ACC) to the top of the central column in situ leads to the production of radiolabeled ethylene by all the flower parts. In addition, the ethylene production of isolated lips, measured immediately after excision, was initially high but ceased within 10 to 15 minutes. Treatment of the central column in situ with ethylene or ethephon did not stimulate ACC production but did stimulate lip coloration and this was accompanied by an increased internal ethylene concentration in the lip. The data indicate that endogenously produced as well as applied ACC is rapidly translocated from the site of production or application to all the other flower parts where it is immediately converted into ethylene. By excision of a flower organ, the influx of ACC is prevented, causing a rapid decrease in ethylene production. In addition, it was found that ethylene may also be translocated in physiologically significant amounts within the flower. The roles of ACC and ethylene as mobile senescence or wilting factors in emasculation- and pollination-induced senescence is discussed.

Use of a laser-driven photoacoustic detection system for measurement of ethylene production in cymbidium flowers.

A laser-based photoacoustic method was used for determination of ethylene (C(2)H(4)) production of emasculated orchid (Cymbidium) flowers in a flow-through system. The laser photoacoustic equipment consisted of a line-tuneable CO(2) laser in conjunction with a single-pass resonant acoustic cell. The minimum detection limit of the system for C(2)H(4) in air was 0.03 nanoliter per liter. C(2)H(4) production of intact Cymbidium (cv Mary Pinchess ;Del Rey') flowers was very low (0.015 nanoliter per gram per hour) and showed an increase within 3 hours following emasculation (removal of pollinia plus anthercap). Production peaked (0.14 nanoliter per gram per hour) 8 hours after emasculation and decreased thereafter. Production again increased 45 hours after emasculation. Coloration of the labellum appeared shortly after the first peak; wilting of the petals and sepals appeared during the second rise in ethylene production. The use of the laser photoacoustic technique in plant physiological studies is discussed.