Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Recognition of Verticillium effector Ave1 by tomato immune receptor Ve1 mediates Verticillium resistance in diverse plant species
Song, Yin - \ 2017
University. Promotor(en): Bart Thomma; Pierre de Wit. - Wageningen : Wageningen University - ISBN 9789463437950 - 231
disease resistance - defence mechanisms - immunity - plant-microbe interactions - plant pathogens - verticillium dahliae - verticillium - tomatoes - solanum lycopersicum - receptors - genes - tobacco - nicotiana glutinosa - potatoes - solanum tuberosum - solanum torvum - humulus lupulus - cotton - gossypium hirsutum - transgenic plants - arabidopsis thaliana - ziekteresistentie - verdedigingsmechanismen - immuniteit - plant-microbe interacties - plantenziekteverwekkers - tomaten - receptoren - genen - tabak - aardappelen - katoen - transgene planten

Plant-pathogenic microbes secrete effector molecules to establish disease on their hosts, whereas plants in turn employ immune receptors to try and intercept such effectors in order to prevent pathogen colonization. Based on structure and subcellular location, immune receptors fall into two major classes; cell surface-localized receptors that comprise receptor kinases (RKs) and receptor-like proteins (RLPs) that monitor the extracellular space, and cytoplasm-localized nucleotide-binding domain leucine-rich repeat receptors (NLRs) that survey the intracellular environment. Race-specific resistance to Verticillium wilt in tomato (Solanum lycopersicum) is governed by the tomato extracellular leucine-rich repeat (eLRR)-containing RLP-type cell surface receptor Ve1 upon recognition of the effector protein Ave1 that is secreted by race 1 strains of the soil-borne vascular wilt Verticillium dahliae. Homologues of V. dahliae Ave1 (VdAve1) are found in plants and in a number of plant pathogenic microbes, and some of these VdAve1 homologues are recognized by tomato Ve1. The research presented in this thesis aims to characterize the role of the tomato cell surface-localized immune receptor Ve1, and its homologues in other diverse plant species, in Verticillium wilt resistance.

Exploring the resistance against root parasitic plants in Arabidopsis and tomato
Cheng, Xi - \ 2017
University. Promotor(en): H.J. Bouwmeester, co-promotor(en): Carolien Ruyter-Spira. - Wageningen : Wageningen University - ISBN 9789463437004 - 305
plants - parasitic plants - arabidopsis thaliana - solanum lycopersicum - host parasite relationships - plant growth regulators - resistance - planten - parasitaire planten - gastheer parasiet relaties - plantengroeiregulatoren - weerstand
Root parasitic plant species such as broomrapes (Orobanche and Phelipanche spp.) and witchweeds (Striga spp.) are notorious agricultural weeds. They cause damage to crops by depriving them of water, nutrients and assimilates via a vascular connection. The difficulty in controlling root parasitic weeds is largely due to their intricate lifecycle and partially underground lifestyle. Their life cycle includes processes such as germination of the seed, the formation of the vascular connection with the host, the growth and development of the parasite after attachment and the emergence of shoots and flowers aboveground. The germination of many parasitic plants is induced by strigolactones that were recently shown to also be signalling compounds that stimulate mycorrhizal symbiosis. In addition, in the past few years, their role in plant development and plant defense has been established revealing them as a new class of plant hormones that exert their function likely in interaction with other hormones.
Prospects of whole-genome sequence data in animal and plant breeding
Binsbergen, Rianne van - \ 2017
University. Promotor(en): Roel Veerkamp; Fred van Eeuwijk, co-promotor(en): Mario Calus. - Wageningen : Wageningen University - ISBN 9789463431903 - 220
next generation sequencing - dna sequencing - quantitative trait loci - cattle - genomics - solanum lycopersicum - animal breeding - plant breeding - dna-sequencing - loci voor kwantitatief kenmerk - rundvee - genomica - dierveredeling - plantenveredeling

The rapid decrease in costs of DNA sequencing implies that whole-genome sequence data will be widely available in the coming few years. Whole-genome sequence data includes all base-pairs on the genome that show variation in the sequenced population. Consequently, it is assumed that the causal mutations (e.g. quantitative trait loci; QTL) are included, which allows testing a given trait directly for association with a QTL, and might lead to discovery of new QTL or higher accuracies in genomic predictions compared to currently available marker panels. The main aim of this thesis was to investigate the benefits of using whole-genome sequence data in breeding of animals and plants compared to currently available marker panels. First the potential and benefits of using whole-genome sequence data were studied in (dairy) cattle. Accuracy of genotype imputation to whole-genome sequence data was generally high, depending on the used marker panel. In contrast to the expectations, genomic prediction showed no advantage of using whole-genome sequence data compared to a high density marker panel. Thereafter, the use of whole-genome sequence data for QTL detection in tomato (S. Lycopersicum) was studied. In a recombinant inbred line (RIL) population, more QTL were found when using sequence data compared to a marker panel, while increasing marker density was not expected to provide additional power to detect QTL. Next to the RIL population, also in an association panel it was shown that, even with limited imputation accuracy, the power of a genome-wide association study can be improved by using whole-genome sequence data. For successful application of whole-genome sequence data in animals or plants, genotype imputation will remain important to obtain accurate sequence data for all individuals in a cost effective way. Sequence data will increase the power of QTL detection in RIL populations, association panels or outbred populations. Added value of whole-genome sequence data in genomic prediction will be limited, unless more information is known about the biological background of traits and functional annotations of DNA. Also statistical models that incorporate this information and that can efficiently handle large datasets have to be developed.

Identification of metabolites involved in heat stress response in different tomato genotypes
Paupière, Marine J. - \ 2017
University. Promotor(en): Richard Visser, co-promotor(en): Arnaud Bovy; Yury Tikunov. - Wageningen : Wageningen University - ISBN 9789463431842 - 168
solanum lycopersicum - tomatoes - genotypes - heat stress - heat tolerance - pollen - metabolomes - metabolites - metabolomics - tomaten - genotypen - warmtestress - hittetolerantie - stuifmeel - metabolomen - metabolieten - metabolomica

Tomato production is threatened by climate change. High temperatures lead to a decrease of fruit set which correlates with a decrease of pollen fertility. The low viability of tomato pollen under heat stress was previously shown to be associated with alterations in specific metabolites. In this thesis, we used untargeted metabolomics approaches to broaden the identification of metabolites affected by heat stress. We assessed the suitability of pollen isolation methods for metabolomics analysis and considered the pitfalls for our further analysis. We explored the developmental metabolomes of pollen and anthers of different tomato genotypes under control and high temperature conditions and identified that microsporogenesis is a critical developmental stage for the production of mature and fertile pollen grain under heat stress. Several metabolites were putatively associated with tolerance to high temperature such as specific flavonoids, polyamines and alkaloids. These metabolites can be further used as markers in breeding programs to develop new genotypes tolerant to high temperatures.

Optimization of productivity and quality of irrigated tomato (Solanum lycopersicum L.) by smallholder farmers in the Central Rift Valley area of Oromia, Ethiopia
Gemechis, Ambecha O. - \ 2017
University. Promotor(en): Paul Struik, co-promotor(en): B. Emana. - Wageningen : Wageningen University - ISBN 9789463431576 - 262
solanum lycopersicum - irrigation - crop production - optimization - photosynthesis - chlorophyll - gas exchange - water use efficiency - crop yield - ethiopia - irrigatie - gewasproductie - optimalisatie - fotosynthese - chlorofyl - gasuitwisseling - watergebruiksrendement - gewasopbrengst - ethiopië

Tomato (Solanum lycopersicum L.) is a vegetable crop with high potential to contribute to poverty reduction via increased income and food security. It is widely grown by smallholders, has high productivity and its demand is increasing. Ethiopia produced about 30,700 Mg of tomatoes on 5,027 ha annually in 2014/2015. Average yields are only 6.1 Mg ha-1, below the world average yields. There is both a need and a potential to increase tomato production per unit area.

The aim of this thesis is to analyze the irrigated tomato production systems of smallholder farmers in Ethiopia, to survey and characterize the tomato in selected ecoregions and seasons, and to identify yield-limiting or yield-reducing factors and opportunities to enhance yield by using a combination of surveys and field experiments. Field experiments on optimization of yield and quality of field-grown tomato were carried out at Ziway, Ethiopia, for two seasons to study the impact of different irrigation practices applied, based on local empirical practices, deficit irrigation, or crop water requirement.

This thesis begins with a survey of tomato production systems. The survey details the area and production in various zones and for each of these zones yield- determining, yield-limiting, and yield-reducing factors and opportunities for improving yield and quality are indicated. It also avails area, production and yield data for each growing season and typifies the production systems in these zones. Low temperature (cold) from October-January and shortage of improved seeds are recognized as yield-determining factors, whereas insufficient water and nutrient (fertilizer) supply proved to be yield-limiting factors across zones. Late blight (Phytophthora infestans), Fusarium wilt (Fusarium oxysporum) and different pests and weeds are identified as yield-reducing factors in the zones. Experienced growers who have access to extension service recorded significant yield increment. Farmers Research Groups improved actual average yield with the use of improved technology (improved varieties and quality seed), and better efficiencies of water and fertilizer use. This study quantified influences of irrigation systems and strategies on growth-determining tomato features. Variation in irrigation systems and strategies accounted for variation in growth and dry matter accumulation. Greater performance for yield-related traits was obtained with drip irrigation based on crop water requirement for tomato varieties. Examination of plants showed also that local empirical irrigation is responsible for the occurrence of Phytophthora root rot, whereas deficit irrigation proved cause for occurrence of Fusarium wilt (Fusarium oxysporum), blossom end rot and broome rape (Orobanche ramosa) on roots or leaves, stems or fruits.

The experiments on irrigation scheduling with different irrigation systems and strategies gave useful indications on the possibility to improve commercial yield (CY) and water use efficiency. Promising results on CY and agronomical water use efficiency of tomato were achieved with drip irrigation based on crop water requirement, while for the biological water use efficiency higher value was obtained with deficit drip irrigation in both seasons. The findings indicate that the CY was decreased significantly for deficit by 50% in drip irrigation and deficit by 50% in furrow irrigation in both seasons. Mean CY for drip irrigation according to crop water requirement increased by 51% and 56% compared with deficit drip irrigation, whereas furrow irrigation based on crop water requirement increased by 52% and 54% compared with deficit furrow in Experiments 1 and 2, respectively. However, water use efficiency decreased with the increasing water volume.

Simultaneous measurements of rate of photosynthesis based on gas exchange measurements and the thylakoid electron flux based on chlorophyll fluorescence were used to investigate physiological limitations to photosynthesis in leaves of deficit irrigated tomato plants under open field situations. Combined leaf gas exchange/chlorophyll fluorescence measurements differentiated the treatments effectively. Reduction in rate of photosynthesis, stomatal conductance and the maximum quantum efficiency of photosystem II varied across seasons of all varieties, whereas leaf temperature was increased by deficit irrigation in all varieties. Among varieties studied, Miya was found relatively tolerant to deficit irrigation. Stomatal limitation of rate of photosynthesis increased significantly as a result of water stress suggesting a strong influence of the stomatal behaviour.

We also determined the influence of irrigation systems and strategies on water saving and tomato fruit quality. Using deficit drip irrigation was the best management strategy to optimize water use and tomato quality. Fruit dry matter content, acid content and total soluble solids were significantly higher with deficit drip irrigation than with other treatments.

From this thesis it appeared that agro-climatic conditions, access to resources and culture all contribute to the relatively low yields of tomato in the Central Rift Valley of Ethiopia. The thesis also proved that significant advances can be made in yield, quality and resource use efficiency.

Susceptibility genes : an additional source for improved resistance
Sun, Kaile - \ 2017
University. Promotor(en): Richard Visser, co-promotor(en): Evert Jacobsen; Yuling Bai. - Wageningen : Wageningen University - ISBN 9789463431415 - 174
solanum tuberosum - potatoes - solanum lycopersicum - tomatoes - genes - susceptibility - plant pathogenic fungi - phytophthora infestans - disease resistance - plant breeding - aardappelen - tomaten - genen - vatbaarheid - plantenziekteverwekkende schimmels - ziekteresistentie - plantenveredeling

Potato is affected by several diseases. Although, resistance can be obtained by introgression of major resistance genes from wild species, this has rarely been durable. Hence, other sources of resistance are highly needed. New research with a focus on loss of function mutations has led to the identification of disease susceptibility (S) genes in plants. The research in this thesis was aimed at the identification and characterization of potato S genes involved in the interaction with Phytophthora infestans and Botrytis cinerea. We selected 11 Arabidopsis thaliana S genes and silenced their potato orthologs by RNAi in the potato cultivar Desiree. The silencing of six genes resulted in resistance to P. infestans. Moreover, silencing of StDND1 reduced the infection of B. cinerea. Microscopic analysis showed that spore attachment and/or germination of P. infestans and B. cinerea was hampered on the leaf surface of StDND1-silenced potato plants. On StDMR1- and StDMR6-silenced potato plants, hyphal growth of P. infestans was arrested by the hypersensitive response-like cell death. Our results demonstrate that impairment of plant S genes may open a new way for breeding potatoes with resistance to pathogens like P. infestans and B. cinerea.

Susceptibility pays off: insights into the mlo-based powdery mildew resistance
Appiano, Michela - \ 2016
University. Promotor(en): Richard Visser, co-promotor(en): Yuling Bai; Anne-Marie Wolters. - Wageningen : Wageningen University - ISBN 9789462579484 - 265
solanum lycopersicum - tomatoes - disease resistance - susceptibility - oidium neolycopersici - genes - gene expression - genomics - molecular breeding - plant breeding - tomaten - ziekteresistentie - vatbaarheid - genen - genexpressie - genomica - moleculaire veredeling - plantenveredeling

Powdery mildew (PM) is a worldwide-occurring plant disease caused by ascomycete fungi of the order Erysiphales. A conspicuous number of plant species are susceptible to this disease, the occurrence of which is increasing due to the influence of climate change. Symptoms are easy to recognize by the powdery whitish fungal structures growing on the surface of plant organs. Severe infections cause significant losses in crops, such as tomato, cucumber and wheat, as well as in ornamentals, like rose and petunia. Accordingly, breeding crops with a robust immunity to this disease is of great economic importance.

A significant step in this direction was the discovery of mlo (mildew locus o) mutant alleles of the barley HvMlo gene, which are responsible for the non-race specific resistance to the barley PM pathogen, Blumeria graminis f.sp. hordei (Bgh). During the years, this recessively inherited resistance was observed to be durable, contrary to the short life-span of resistances conferred by dominant resistance (R-) genes used in barley breeding programs. Studies on the histological mechanisms of the mlo-based resistance showed that the PM pathogen was stopped during penetration of the cell wall by the formation of a papilla. This structure prevents the formation of the feeding structure of the pathogen, called a haustorium.

After sequencing many plant genomes, we are discovering that MLO genes are not only typical of this cereal, but are ubiquitously present in higher plant species in multiple copies per species, forming a gene family. The impairment of some members of a number of ever increasing plant species lead to broad-spectrum resistance towards their adapted PM pathogens. For example, in tomato the ol-2 gene, naturally harbored by the cherry tomato Solanum lycopersicum var. cerasiforme, represents the loss-of-function allele of the SlMLO1 gene, conferring resistance to the PM pathogen Oidium neolycopersici (On). Consequently, the use of mlo mutants represents a suitable alternative to the classical use of R-genes in breeding programs.

In Chapter 2, we describe the in silico identification of the complete tomato SlMLO gene family using the available information in the SOL genomic network database. In total, 16 tomato SlMLO members were cloned from leaf, root, flower and fruit of the susceptible tomato cv. Moneymaker to confirm the sequences retrieved from the database and to verify their actual expression in these tissues. We observed the presence of various types of splicing variants, although their possible functional meaning has not been investigated. Motif analyses of each of the translated protein sequences and phylogenetic studies highlighted, on one hand, amino acid stretches that characterize the whole MLO family, and, on the other hand, stretches conserved in MLO homologs that are phylogenetically related. Following a gene expression study upon On inoculation, we identified members of the SlMLO family that are upregulated few hours after pathogen challenge. Except SlMLO1, none of the three newly identified homologs in clade V, thus phylogenetically close to SlMLO1, are induced. Interestingly, two homologs, each found in different clades, are upregulated similarly to SlMLO1. Using an RNAi approach, we silenced the additional clade V-SlMLO homologs, namely SlMLO3, SlMLO5 and SlMLO8, to investigate their possible role in PM resistance. We observed that none of these homologs if individually silenced, leads to PM resistance. However, if SlMLO5 and SlMLO8 are silenced together with SlMLO1, a significantly higher level of resistance is achieved compared to plants carrying the ol-2 allele. The role of SlMLO3 could not be verified. We, therefore, concluded that there are three SlMLO genes in tomato unevenly contributing to the PM disease, of which SlMLO1 has a major role.

Chapter 3 focuses on the components of the tomato mlo-based resistance. In Arabidopsis, it is known that four members of the SNARE protein family, involved in membrane fusion, are involved in mlo-based resistance. In this chapter, we focused on the identification of tomato homologs of the Arabidopsis syntaxin PEN1 (AtSYP121). Among the group of syntaxins identified in tomato, two were closely related to each other and also to AtPEN1, denominated SlPEN1a and SlPEN1b. Another Arabidopsis syntaxin that shows a high level of homology with PEN1, called SYP122, was also found to group together with the newly identified SlPEN1 genes. However, the role of SYP122 in plant immunity was not shown in literature. After obtaining individual silencing RNAi constructs, we transformed the resistant ol-2 line, and we challenged the obtained transformants with the adapted PM On, and the non-adapted Bgh. Interestingly, we observed a significant On growth and an enhanced Bgh cell entry only in SlPEN1a silenced plants but not in SlPEN1b silenced ones. We performed a protein alignment of tomato and Arabidopsis functional and non-functional PEN sequences. The presence of three differently conserved non-synonymous amino-acid substitutions is hypothesised to be responsible for the specialization in plant immune function.

In Chapter 4 and Chapter 5, we build up a body of evidence pointing to the fact that the function of the MLO susceptibility genes is highly conserved between monocot and dicot plant species.

In Chapter 4 we started by identifying and functionally characterizing two new MLO genes of Solanaceous crops affected by the PM disease, tobacco (Nicotiana tabacum) and eggplant (Solanum melongena). We named them NtMLO1 and SmMLO1 in the respective species, as they are the closest homologs to tomato SlMLO1. By overexpressing these genes in the resistant ol-2 line, we obtained transgenic plants that were susceptible to the PM pathogen On. This finding demonstrates that both heterologous MLO proteins can rescue the function of the impaired ol-2 allele in tomato. In addition, we found in tobacco NtMLO1 an amino acid (Q198) of critical importance for the susceptibility function of this protein.

In Chapter 5, we used the same approach adopted in Chapter 4 to show that other MLO proteins of more distant dicot species, like pea PsMLO1, can rescue the loss-of-function of the tomato ol-2 allele. And finally, we stretched this concept also to monocot MLO proteins, using barley HvMlo. While performing these experiments, we could verify that the function of the monocot and dicot susceptibility MLO proteins does not rely on the presence of class-specific conservation. The latter can be the reason for the phylogenetic divergence, placing monocot MLO proteins in clade IV and dicot MLO proteins in clade V of the phylogenetic MLO tree. However, functional conservation might depend on crucial shared amino acids of clade IV and V MLO proteins. Therefore, we also conducted a codon-based evolutionary analysis that resulted in the identification of 130 codons under negative selection, thus strongly maintained during evolution.

In Chapter 6 we introduce the PM disease in cucumber caused by Podosphaera xanthii (Px). We cloned the candidate susceptibility gene for PM in cucumber, CsaMLO8, from susceptible and resistant genotypes. The latter was described as an advanced cucumber breeding line characterized by hypocotyl resistance. In this line, we found the presence of aberrant splicing variants of the CsaMLO8 mRNA due to the insertion in its corresponding genomic region of a Class LTR retrotransposon. Heterologous expression of the wild-type cucumber allele in the tomato ol-2 line restored its PM susceptibility, while the heterologous expression of the aberrant protein variant failed to do so. This finding confirms that the resistance of the advanced cucumber breeding line is due to the disruption of the coding region of this gene. We also showed that the expression of CsaMLO8 in the susceptible genotype is induced by Px in hypocotyl tissue, but not in cotyledon or leaf. Finally, by examination of the resequencing data of a collection of 115 cucumber accessions, we found the presence of the TE-containing allele in 31 of them among which a wild cucumber accession that might have been used in breeding programs to obtain resistance to the PM disease in cucumber.

In Chapter 7 a novel loss-of-function allele of the SlMLO1 gene is described, designated m200. This allele was found in a resistant plant (M200) from a mutagenized tomato Micro-Tom (MT) population obtained with the chemical mutagen ethyl methanesulfonate (EMS). The m200 mutation corresponds to a nucleotide transversion (T à A) which results in a premature stop codon. The length of the predicted SlMLO1 protein in the M200 plant is only 21 amino acids, thus much shorter than the predicted protein of the previously described ol-2 allele, consisting of 200 amino acids. Thanks to the development of a High-Resolution Melting (HRM) marker designed to detect the m200 mutation, we observed that this allele confers recessively inherited resistance in backcross populations of the resistant M200 plant with MT and Moneymaker. Histological study showed that the resistance of the m200 mutant is associated with papilla formation. Finally, we compared the rate of On penetration in epidermal cells of m200 plants with the one of plants carrying the ol-2 allele and the transgenic plants in which multiple SlMLO homologs were silenced, generated in Chapter 2.

Ultimately, in Chapter 8 the results of the previous chapters are discussed in the context of 1) practical applications in breeding programs aimed at introducing the mlo-based resistance in new crops, 2) possible research aimed at unraveling the function of the MLO protein and 3) the role of other SNARE proteins.

Het Nieuwe Gewas : sturen van de plantvorm voor verhoogde lichtbenutting
Gelder, Arie de; Janse, Jan ; Warmenhoven, Mary - \ 2016
Bleiswijk : Wageningen UR Glastuinbouw (Rapport GTB 1407) - 62
tomaten - solanum lycopersicum - kasgewassen - glasgroenten - glastuinbouw - energiebesparing - plantenontwikkeling - gewasteelt - licht - tomatoes - greenhouse crops - greenhouse vegetables - greenhouse horticulture - energy saving - plant development - crop management - light
Wageningen UR Greenhouse horticulture searched in the “The New Crop” project for the crop structure that best contributes to the goal of energy-efficient production and therefore energy saving, by removing 33, 44 or 55% of the leaves at a young stage. On October 10th 2014 the experiment started with topped plants of the tomato variety Brioso grafted on Maxifort. Dry matter production was lowest in the very open crop, however partitioning to the fruits was highest in that treatment. Therefore, this crop produced in the winter under assimilation lighting most. In summer, the standard treatment was the best and the production in this treatment was highest. The plants in the very open treatment were shorter and had smaller leaves than the plants of the standard treatment. This is might be due to a different red: far red ratio of light in the crop. Leaf picking at a young stage contributes to better distribution of assimilates to the fruits. This can be used as a crop management measure as the plant in winter develops too much leaves. The project was funded by the Dutch energy transition program “Kas als Energiebron”.
Light on phloem transport (an MRI approach)
Prusova, Alena - \ 2016
University. Promotor(en): Herbert van Amerongen, co-promotor(en): Henk van As. - Wageningen : Wageningen University - ISBN 9789462579156 - 130
solanum lycopersicum - phloem - light - flow - photoperiod - nuclear magnetic resonance - biophysics - magnetic resonance imaging - floëem - licht - stroming - fotoperiode - kernmagnetische resonantie - biofysica - kernspintomografie

This thesis (Light on phloem transport – an MRI approach) aims to answer the question whether phloem transport can be a limiting factor for photosynthesis efficiency (and ultimately causing a bottleneck towards achieving higher yields). To answer this key question, we manipulated the source: sink ratio within tomato (Solanum lycopersicum L.) while measuring phloem transport with magnetic resonance imaging (MRI) flowmetry. Additionally we compared phloem flow characteristics of two potato plants (Solanum tuberosum L.) which differed in source : sink ratio. In Chapter 2, the source strength was manipulated by varying the light intensity. An increase in phloem sap volume flow under higher light intensities was observed. However, under all light intensities applied, the phloem flow velocity was found to be constant (as has previously been suggested in other studies) although a clear diurnal pattern was observed. This finding does not fit in current models to describe the mechanism of phloem transport and a different mechanism must be at play. The results of this chapter demonstrate that increased levels of photo-assimilates are transported in sieve tubes, which are activated when needed by the plant. This is the first study which shows that plants activate individual sieve tubes when more photo-assimilates are available, yet maintain constant velocity. Those observations were in a tomato plant with pruned fruit trusses (i.e., in a simplified system). In Chapter 3, we investigated whether tomato plants still exhibit constant phloem flow velocity (with a diurnal pattern) under normal conditions, i.e., with strong sinks (tomato fruits) still attached. This was tested for both a long and short photoperiod by measuring flow characteristics with MRI flowmetry. We simultaneously monitored other plant processes like xylem flow rates with a heat balance sensor, net photosynthesis with gas exchange and stem diameter changes with a linear motion potentiometer. With this integrated approach, we revealed a correlation between night phloem volume flow, dark respiration and stem growth. We also conclusively showed that phloem volume flow performs a diurnal pattern under a variety of source-sink ratios which appears to be a normal behaviour for tomato plants growing under moderately-high light conditions. In chapters 2 and 3 we learned that under higher source strength a greater amount of phloem sap is transported, but the changes in flow were not accompanied by changes in velocity. To further our understanding of the mechanisms driving phloem transport, it is of interest to know how the sucrose concentration in phloem sap relates to phloem flow. In Chapter 4 we used an average T2 relaxation time in the phloem vascular tissue region to reveal the plant’s phloem carbon status under source manipulation. In this chapter we demonstrated that T2 relaxation time, when measured in parallel with phloem flow, can provide additional information about phloem region carbon status, i.e., changes in the T2 relaxation time are correlated with changes in sucrose concentration in the whole phloem region.

When studying phloem transport in plants with magnetic resonance imaging (MRI) flowmetry, plants which are relatively easy to manipulate (e.g. fruit pruning) like tomato have so far been used. However, tomato plants (used in all three previous chapters) have relatively low sink strength beneath the MRI measurement site. A potentially preferable approach is to work with plants with strong sinks beneath the measurement site. In Chapter 5 we studied potato as a potentially better test subject for MRI flowmetry as it possesses strong sink below the MRI measurement site (i.e., developing tubers). For that purpose we used two potato plants (cv. Desiree) both with several developing tubers. One of the plants overexpressed the StSWEET gene (35S:StSWEET) which appears to have altered its source : sink ratio. As a result, the 35S:StSWEET plant transported 60% more phloem sap than Desiree WT. Strikingly, the average phloem flow velocity in both plants was the same and the greater amount of transported phloem sap in the 35S:StSWEET plant was accommodated by more sieve tubes than in Desiree WT. This finding agrees with the hypothesis about the conserved nature of phloem flow velocity, where volume flow is regulated by the number of active sieve tubes (Chapter 2 and 3). In this chapter we also demonstrate that a potato plant with developing tubers represents a good subject to study phloem transport with MRI flowmetry. We concluded that under optimal conditions (which are commonly met in greenhouses) phloem transport is likely to reach its maximum capacity and therefore photosynthesis could be limited by the export and transport of photo-assimilates because of the finite number of sieve tubes and constant flow velocity.

Plantmonitoring op basis van fotosynthese sensoren : ontwikkelen en testen van sensoren
Dieleman, Anja ; Bontsema, Jan ; Jalink, Henk ; Snel, Jan ; Kempkes, Frank ; Voogt, Jan ; Pot, Sander ; Elings, Anne ; Jalink, Vincent ; Meinen, Esther - \ 2016
Bleiswijk : Wageningen UR Glastuinbouw (Rapport GTB 1405) - 86
teelt onder bescherming - glastuinbouw - kastechniek - sensors - fotosynthese - kooldioxide - energie - energiebesparing - verlichting - kunstlicht - kunstmatige verlichting - ventilatie - kunstmatige ventilatie - fluorescentie - tomaten - solanum lycopersicum - protected cultivation - greenhouse horticulture - greenhouse technology - photosynthesis - carbon dioxide - energy - energy saving - lighting - artificial light - artificial lighting - ventilation - artificial ventilation - fluorescence - tomatoes
The basic process for crop growth and production is photosynthesis. Measuring crop photosynthesis is therefore important to monitor the status of the crop and whether the greenhouse climate is set to the needs of the crop. In this project, two monitoring systems for crop photosynthesis were developed and tested. (1) The crop photosynthesis monitor is a soft sensor that can calculate the CO2 uptake of an entire crop. The basis for these calculations are the balance between CO2 supply and CO2 loss via ventilation and crop photosynthesis. By measuring the CO2 concentration and humidity inside and outside the greenhouse, the crop photosynthesis can be calculated. (2) The CropObserver is a fluorescence sensor that measures the light use efficiency of photosynthesis of a large crop area (3 x 3 m2). The crop receives light pulses from a laser in the top of the greenhouse, the sensor measures the fluorescence signal of the crop. Both sensors were tested in a tomato crop in 2014 with promising results. The sensors functioned without problems and delivered patterns of daily photosynthesis which matched the reference measurements reasonably well up to well.
2SaveEnergy-Gewächshaus- Produktion und Energieverbrauch
Kempkes, F.L.K. ; Janse, J. - \ 2016
Bleiswijk : Wageningen UR Glastuinbouw (Rapport GTB 1404) - 46 p.
greenhouse horticulture - greenhouse crops - greenhouse technology - energy saving - energy consumption - isolation - isolation techniques - tomatoes - solanum lycopersicum - glastuinbouw - kasgewassen - kastechniek - energiebesparing - energiegebruik - isolatie - isolatietechnieken - tomaten
Energy savings through a greenhouse cover of insulation glass requires a large investment. In the search for a cheaper alternative by a consortium of companies consisting of VDH Plastic Greenhouses, Van der Valk Horti Systems, AGC Chemicals Europe en Boal Systems a Glass-Film-greenhouse cover, better known as the 2SaveEnergy greenhouse concept was realized in summer 2014. The combination of clear glass with a diffuse ETFE film and a double screen mounted at a distance of only a few centimetres, in the year 2015, resulted in a low energy consumption and a better than expected good tomato production. During cultivation, the principles of the new cultivation methods were used. With respect to the common practice, the energy consumption was more than 50% lower at a minimum equal production.
Teelt en energie 2SaveEnergy kas
Kempkes, F.L.K. ; Janse, J. - \ 2016
Bleiswijk : Wageningen UR Glastuinbouw (Rapport GTB 1402) - 42 p.
glastuinbouw - kasgewassen - kastechniek - energiebesparing - energiegebruik - isolatie - isolatietechnieken - tomaten - solanum lycopersicum - greenhouse horticulture - greenhouse crops - greenhouse technology - energy saving - energy consumption - isolation - isolation techniques - tomatoes
Energy savings through a greenhouse cover of insulation glass requires a large investment. In the search for a cheaper alternative by a consortium of companies consisting of VDH Plastic Greenhouses, Van der Valk Horti Systems, AGC Chemicals Europe en Boal Systems a Glass-Film-greenhouse cover, better known as the 2SaveEnergy greenhouse concept was realized in summer 2014. The combination of clear glass with a diffuse ETFE film and a double screen mounted at a distance of only a few centimetres, in the year 2015, resulted in a low energy consumption and a better than expected good tomato production. During cultivation, the principles of the new cultivation methods were used. With respect to the common practice, the energy consumption was more than 50% lower at a minimum equal production.
Heat stress tolerance responses in developing tomato anthers
Bita, Elena - \ 2016
University. Promotor(en): Gerco Angenent, co-promotor(en): Christian Bachem. - Wageningen : Wageningen University - ISBN 9789462577701 - 109 p.
tomatoes - solanum lycopersicum - anthers - heat stress - stress tolerance - heat tolerance - heat shock - transcriptomics - reproductive performance - gene expression profiling - meiosis - tomaten - helmknoppen - warmtestress - stresstolerantie - hittetolerantie - hitteshock - transcriptomica - voortplantingsvermogen - genexpressieprofilering - meiose

Global warming already has and will significantly impact crop productivity and yield in the near future. In order to meet the forecasted requirements of the future agricultural production, a proper assessment of crops environmental stress tolerance needs to be designed and implemented, from the laboratory to field. Genetic variation in the ability of tomatoes to set fruit under high temperature conditions has made selection for heat tolerance possible and multiple opportunities for improvement exist, as tolerance to high temperatures is a multi-genic character involving a complex network of chaperones and other protective proteins acting together to defend the cells from heat injury. Breeding programs involved in the development of heat tolerant cultivars should identify and make use of such tolerance traits already available in collected or wild germplasm.

The goal of this thesis was to characterize the response to high temperatures in meiotic tomato anthers with contrasting responses to heat and to identify genes that could be related to thermo-tolerance mechanisms during gamete development. Several molecular tools such as transcriptomic profiling by cDNA-AFLP and microarray analysis, RT-PCR or in situ RNA hybridisation were used to achieve this goal.

The second chapter reviews the effects of heat stress on reproductive flower development, candidate tolerance pathways and methods for production of heat tolerant crops.

The third chapter provides a general overview of the expression changes occurring in the developing anthers of a sensitive tomato genotype following exposure to a (short and) moderate high temperature stress (MHS). Using a combination of cDNA-AFLP, RT-PCR, and in situ RNA hybridisation, we characterized and verified the general transcriptional response to heat of tomato plants. Our results revealed that approximately 1% of the examined transcript-derived fragments exhibit alterations in expression pattern and the majority of these were down-regulated The putative functions associated with the genes identified by cDNA-AFLP indicated involvement of heat shock, metabolism, antioxidant and developmental processes. Based upon the observed transcriptional changes in response to MHS and on literature sources, we identified a number of candidate transcripts to be involved in heat-tolerance. The spatial expression of several such candidate genes was further examined using in situ RNA hybridisation and this showed that the investigated genes are expressed in the tapetum or/and in developing microspores. Furthermore, the expression of several candidate genes has been quantified by RT-PCR in additional genotypes with different degrees of heat tolerance. The results suggested a correlation between gene expression levels, pollen germination rates and tolerance to heat (Chapter 4).

In the fourth chapter we proceeded to profile the response to heat of meiotic anthers in a tolerant and a sensitive tomato genotype and investigated the expression of the identified candidate genes in several pairs of contrasting genotypes. Using microarray analysis (for an extensive overview of the meiotic response to heat) and RT-PCR, we were able to clearly distinguish differential responses of the tolerant genotype. After 2h of moderate heat stress, the heat-tolerant genotype exhibits fewer transcriptional changes than the heat-sensitive genotype. In the heat-tolerant genotype, the majority of changes in gene expression is represented by up-regulation, while in the heat-sensitive genotype there is a general trend to down-regulate gene expression soon after MHS. Moreover, the heat-tolerant genotype also shows a different level of constitutive gene expression profiles when compared to the heat-sensitive genotype indicating a difference in genetic adaptation with regards to increased temperatures. The putative functions associated with the genes identified by microarray profiling indicate involvement of heat shock, antioxidant, metabolic, and cell development pathways. Based upon the observed differences in response to MHS we selected a number of candidate transcripts involved in heat-tolerance and confirmed their expression pattern in different tomato genotypes with contrasting responses to heat. The results suggested that the candidate genes are involved in the activation of protection mechanisms in the tomato anthers during moderate heat stress and, could therefore contribute to normal growth and development of the male gametophyte and implicitly a successful fruit set under adverse temperatures.

In the fifth chapter we tested the hypothesis that heat tolerance is associated with maintenance of organ identity, fertility and lower ABA levels during heat stress (for several tomato genotypes) and analysed the dynamics of ABA accumulation under temperature stress in several tomato genotypes with contrasting responses to heat. Furthermore, pollen germination tests were performed and additional physiological aspects of anther development for each genotype were analysed as well. The general trend observed was the accumulation of lower relative levels of ABA at the end of the experimental period compared to the initial stages in more tolerant genotypes and of higher levels in the sensitive genotypes. We concluded from these results that the morphological changes in the floral tissues and the overall changes in ABA levels are correlated with the molecular responses under increased temperature in the genotypes analysed. Whether these correlations are causally related is not clear; therefore more research is needed to resolve these issues.

The sixth chapter examines our analysis of the heat stress response in meiotic tomato anthers in a broader scientific context. I discuss the different aspects of our results and present several candidate genes involved in plant thermo-tolerance. In addition, I also discuss the potential involvement of plant growth regulators in plants´ responses to heat stress and suggest various potential follow-up experimental strategies.

Leaf anatomy and photosynthesis : unravelling the CO2 diffusion pathway in C3 leaves
Berghuijs, H.N.C. - \ 2016
University; KU Leuven. Promotor(en): Paul Struik; Bart M. Nicolaï, co-promotor(en): Xinyou Yin. - Wageningen : Wageningen University - ISBN 9789462577947 - 286 p.
leaves - plant anatomy - photosynthesis - mesophyll - photorespiration - carbon pathways - solanum lycopersicum - bladeren - plantenanatomie - fotosynthese - bladmoes - fotorespiratie - koolstofpathways

Keywords: CO2 diffusion, C3 photosynthesis, mesophyll conductance, mesophyll resistance, re-assimilation, photorespiration, respiration, tomato

Herman Nicolaas Cornelis Berghuijs (2016). Leaf anatomy and photosynthesis; unravelling the CO2 diffusion pathway in C3 leaves. PhD thesis. Wageningen University, Wageningen, The Netherlands, with summaries in English and Dutch. 286 pages

Optimizing photosynthesis can contribute to improving crop yield, which is necessary to meet the increasing global demand for food, fibre, and bioenergy. One way to optimize photosynthesis in C3 plants is to enhance the efficiency of CO2 transport from the intercellular air space to Rubisco. The drawdown of CO2 between these locations is commonly modelled by Fick's first law of diffusion. This law states that the flux from the air spaces to Rubisco is proportional to the difference in partial pressure between these locations. The proportionality constant is the mesophyll conductance. Its inverse is mesophyll resistance. Mesophyll resistance is a complex trait, which lumps various structural barriers for CO2 transport and processes that add or remove CO2 along the diffusion pathway. In order to better understand how and to what extent these factors affect photosynthesis, it is necessary to find a more mechanistic description of CO2 transport in the mesophyll. The aim of this dissertation is to investigate how leaf anatomical properties and CO2 sources and sinks along the CO2­ diffusion pathway in C3 leaves affect the photosynthetic capacity of these leaves. In this study, Solanum lycopersicum was used as a model organism. In a first approach, we developed a model in which we partitioned mesophyll resistance into two sub-resistances. The model assumed that CO2 produced by respiration and photorespiration was released between the two sub-resistance components. By quantifying these resistances using measured thicknesses, exposed mesophyll and chloroplast surfaces, and assumed diffusive properties, we were able to simulate the effect of various anatomical properties on photosynthesis. A disadvantage of this two-resistance approach is that it assumes either that (photo)respiratory CO2 release takes place in the outer cytosol or that there is no CO2 gradient in the cytosol. Therefore, in a second approach we modelled CO2 transport, production and consumption by use of a reaction-diffusion model. This model is more flexible in terms of determining the location of CO2 sources and sinks. We developed methods to estimate physiological parameters of this model using combined gas exchange and chlorophyll fluorescence measurements on leaves. The results suggest that the rate of respiration depends on the oxygen partial pressure, which is often not considered in previous photosynthesis models. We also presented a method to calculate the fraction of (photo)respiratory CO2 that is re-assimilated. We found that this fraction strongly depends on both environmental factors (CO2, irradiance), the location of mitochondria relative to the chloroplast, stomatal conductance and various physiological parameters. The reaction-diffusion model and associated methods presented in this study provide a more mechanistic framework to describe the CO2 diffusion pathway in C3 leaves. This model could, therefore, contribute to identifying targets to increase mesophyll conductance in future research.

Supplementary data: Combined biotic and abiotic stress resistance in tomato
Kissoudis, C. ; Chowdhury, Rawnaq ; Heusden, A.W. van; Wiel, C.C.M. van de; Finkers, H.J. ; Visser, R.G.F. ; Bai, Y. ; Linden, C.G. van der - \ 2016
solanum lycopersicum - tomatoes - disease resistance - stress tolerance - defence mechanisms - plant diseases - abiotic injuries - stress response - phenotypic variation - genetic analysisplant breeding - salt tolerance
Abiotic and biotic stress factors are the major constrains for the realization of crop yield potential. As climate change progresses, the spread and intensity of abiotic as well as biotic stressors is expected to increase, with increased probability of crops being exposed to both types of stress. Shielding crops from combinatorial stress requires a better understanding of the plant’s response and its genetic architecture. In this study, we evaluated resistance to salt stress, powdery mildew and to both stresses combined in tomato, using the S. habrochaites LYC4 introgression line (IL) population. The IL population segregated for both salt stress tolerance and powdery mildew resistance. Using SNP array marker data, QTLs were identified for salt tolerance as well as Na+ and Cl- accumulation. Salt stress increased the susceptibility of the population to powdery mildew in an additive manner. Phenotypic variation for disease resistance was reduced under combined stress as indicated by the coefficient of variation (CV). No correlation was found between disease resistance and Na+ and Cl- accumulation under combined stress Most genetic loci were specific for either salt stress tolerance or powdery mildew resistance. These findings increase our understanding of the genetic regulation of responses to abiotic and biotic stress combinations and can provide leads to more efficiently breeding for tomatoes and other crops with a high level of disease resistance while maintaining their performance in combination with abiotic stress.
Supplementary data: Responses to combined abiotic and biotic stress in tomato are governed by stress intensity and mechanism of resistance
Kissoudis, C. ; Sri Sunarti, Sri ; Wiel, C.C.M. van de; Visser, R.G.F. ; Linden, C.G. van der; Bai, Y. - \ 2016
solanum lycopersicum - tomatoes - disease resistance - stress tolerance - defence mechanisms - plant diseases - abiotic injuries - stress response - phenotypic variation - genetic analysisplant breeding - salt tolerance
Stress conditions in agricultural ecosystems can occur in variable intensities. Different resistance mechanisms to abiotic stress and pathogens are deployed by plants. Thus, it is important to examine plant responses to stress combinations under different scenarios. Here, we evaluated the effect of different levels of salt stress ranging from mild to severe (50, 100 and 150mM NaCl) on powdery mildew (PM) resistance and overall performance of tomato introgression lines with contrasting levels of partial resistance, as well as isogenic lines carrying the PM resistance genes Ol-1 (associated with slow Hypersensitivity Response; HR), ol-2 (a mlo mutant associated with papilla formation) and Ol-4 (a R gene associated with fast HR). PM resistance was affected by salt stress in a genotype and stress intensity dependent manner. In susceptible and partial resistant lines, increased susceptibility was observed under mild salt stress (50mM) which was accompanied with accelerated cell death-like senescence. On the contrary, severe salt stress (150mM) reduced disease symptoms. Na+ and Cl- accumulation in the leaves was linearly related to the decreased pathogen growth under severe stress, suggesting a more direct role for the salt in suppressing PM growth. In contrast, complete resistance mediated by ol-2 and Ol-4 was unaffected under all treatment combinations, and was associated with a decreased growth penalty. Increased susceptibility and senescence under combined stress of the variety Moneymaker (MM) and the NIL Ol-1 was associated with the induction of ethylene and jasmonic acid pathway genes as well as of the cell wall invertase gene LIN6. These results highlight the significance of stress severity and resistance type on the plant’s performance under abiotic and biotic stress combination.
Supplementary data: Hormone signalling regulation of tomato response to combined biotic and abiotic stress
Kissoudis, C. ; Sri Sunarti, Sri ; Wiel, C.C.M. van de; Visser, R.G.F. ; Linden, C.G. van der; Bai, Y. - \ 2016
solanum lycopersicum - tomatoes - disease resistance - stress tolerance - defence mechanisms - plant diseases - abiotic injuries - stress response - phenotypic variation - genetic analysisplant breeding - salt tolerance
Plant hormones are paramount to plant adaptation to changing environmental conditions and interactions with microorganisms. There is currently limited knowledge on their significance in the response to stress combination. Using near isogenic lines (NILs) that carry the Ol-1, ol-2 and Ol-4 gene for resistance to tomato powdery mildew caused by Oidium neolycopersici, this study focused on the responses of these NILs to powdery mildew and salt stress combination. In these NILs, marker genes for monitoring hormonal pathways showed differential expression pattern upon powdery mildew infection. Further by crossing these NILs with tomato mutants notabilis (ABA-deficient), defenseless1 (JA-deficient) and epinastic (ET overproducer) the cross-talk among hormonal pathways was further investigated. Among the mutants, epinastic resulted in increased susceptibility of NIL-Ol-1 and breakdown of NIL-ol-2 resistance, accompanied by reduced callose deposition, effects that were more pronounced under combination with salt stress. On the other hand notabilis, resulting in H2O2 overproduction greatly reduced susceptibility of NIL-Ol-1 under combined stress accompanied however by heightened sensitivity to salt stress. Callose deposition reduction led to partial resistance breakdown in NIL-ol-2 which was reversed under combined stress. NIL-Ol-4 resistance remained robust across all mutant and treatment combinations. We discuss the critical role that hormone signalling appears to have for the outcome of combined stress and powdery mildew in terms of resistance and plant fitness integrating observations from physiological, histochemical and gene expression analyses. These significant insights obtained extend our understanding of hormonal regulation of combined stress responses and can aid in narrowing down targets for improving crop performance under stress combinations.
Supplementary data: Roles and contribution of tomato WRKY genes to salt stress and powdery mildew resistance
Kissoudis, C. ; Gao, D. ; Pramanik, Dewi ; Birhanu, Mengistu ; Wiel, C.C.M. van de; Visser, R.G.F. ; Bai, Y. ; Linden, C.G. van der - \ 2016
solanum lycopersicum - tomatoes - disease resistance - stress tolerance - defence mechanisms - plant diseases - abiotic injuries - stress response - phenotypic variation - genetic analysisplant breeding - salt tolerance
WRKY is a transcription factor family unique to plants with diverse functions in defense pathways, abiotic stress tolerance and developmental programs. Family members are characterized by the conserved WRKY domain and significant sequence variation in the remainder of the protein, which is translated into distinct functions even for closely related genes. We utilized the extensive functional characterization of the Arabidopsis thaliana WRKY family to identify tomato homologues of Arabidopsis WRKY genes that are involved in defense responses (AtWRKY 11, 29, 48, 70 and 72). In total 13 tomato WRKY homologues were identified for these genes, of which 9 were successfully over-expressed, and 12 stably silenced via RNAi in transgenic tomato lines. The transgenic lines were evaluated for their response to salt stress, powdery mildew resistance and the combination of these stresses. Lines overexpressing SlWRKY11 and SlWRKY23, and RNAi lines of SlWRKY7 and SlWRKY9 showed both increased biomass and improved salt tolerance. For SlWRKY11 and SlWRKY23 overexpression (OE) lines, this was accompanied by a moderate increase in oxidative stress tolerance. The SlWRKY6-OE line showed strongly improved salt stress tolerance, but a growth penalty under control conditions. Exceptional phenotypes were observed for the SlWRKY10-OE line (stunted growth) and the RNAi line SlWRKY23-RNAi (necrotic symptoms), but these phenotypes were partly restored to normal under salt stress. Both these lines exhibited increased resistance to powdery mildew, but this was compromised when the plants were put under salt-stress as well. Important functions for tomato WRKY genes were revealed in both the abiotic and biotic stress response and several genes should be further explored to elucidate their downstream regulatory functions that lead to increased stress tolerance.
Environmental and physiological control of dynamic photosynthesis
Kaiser, M.E. - \ 2016
University. Promotor(en): Leo Marcelis, co-promotor(en): Jeremy Harbinson; Ep Heuvelink. - Wageningen : Wageningen University - ISBN 9789462576346 - 248 p.
solanum lycopersicum - arabidopsis thaliana - photosynthesis - carbon dioxide - temperature - humidity - light intensity - fotosynthese - kooldioxide - temperatuur - vochtigheid - lichtsterkte

Irradiance is the main driver of photosynthesis. In natural conditions, irradiance incident on a leaf often fluctuates, due to the movement of leaves, clouds and the sun. These fluctuations force photosynthesis to respond dynamically, however with delays that are subject to rate constants of underlying processes, such as regulation of electron transport, activation states of enzymes in the Calvin cycle, and stomatal conductance (gs). For example, in leaves adapted to low irradiance that are suddenly exposed to high irradiance, photosynthesis increases slowly (within tens of minutes); this process is called photosynthetic induction. Photosynthesis in fluctuating irradiance (dynamic photosynthesis) is limited by several physiological processes, and is further modulated by environmental factors other than irradiance, such as CO2 concentration, air humidity and temperature. Studying dynamic photosynthesis and its environmental and physiological control can help to identify targets for improvements of crop growth, improve the accuracy of mathematical models of photosynthesis, and explore new, dynamic lighting strategies in greenhouses.

In this thesis, the limitations acting on dynamic photosynthesis are explored by reviewing the literature, by experimenting with a suite of environmental factors (CO2 concentration, temperature, air humidity, irradiance intensity and spectrum), genetic diversity in the form of mutants, genetic transformants and ecotypes, and by mathematical modelling. Several genotypes of tomato (Solanum lycopersicum) and the model plant Arabidopsis thaliana, all grown in climate chambers, were used in the experiments. The main findings of the thesis are that a) CO2 concentration and air humidity strongly affect the rate of change of dynamic photosynthesis through a combination of diffusional and biochemical limitations; b) Rubisco activation kinetics are pivotal in controlling rates of photosynthesis increase after a stepwise increase in irradiance, and are further affected by CO2 concentration; c) gs limits photosynthetic induction kinetics in A. thaliana but not in tomato in ambient conditions, and becomes a stronger limitation in low CO2 concentration or air humidity; and d) mesophyll conductance, non-photochemical quenching (NPQ) and sucrose synthesis do not limit dynamic photosynthesis under the conditions used.

In Chapter 1, the rationale for the research conducted is described, by introducing the concept of fluctuating irradiance and its effects on photosynthesis rates. The chapter discusses how dynamic photosynthesis is measured and described, and provides a range of possible applications of the insights gained by the research conducted in this dissertation.

In Chapter 2, the current literature is reviewed and a mechanistic framework is built to explore the effects that the environmental factors CO2 concentration, temperature and air humidity have on rates of dynamic photosynthesis. Across data from literature, higher CO2 concentration and temperature speed up photosynthetic induction and slow down its loss, thereby facilitating higher rates of dynamic photosynthesis. Major knowledge gaps exist regarding the loss of photosynthetic induction in low irradiance, dynamic changes in mesophyll conductance, and the extent of limitations imposed by gs across species and environmental conditions.

Chapter 3 is an experimental exploration of the effects of CO2 concentration, leaf temperature, air humidity and percentage of blue irradiance on rates of photosynthetic induction in dark-adapted tomato leaves. Rubisco activation, changes in stomatal and mesophyll conductance, diffusional and biochemical limitations, efficiency of electron transport through photosystem II, NPQ and transient water use efficiency, were examined to give a comprehensive overview of the environmental modulation of dynamic photosynthesis. Unlike the percentage of blue irradiance, increases in CO2 concentration, leaf temperature and air humidity all positively affected the rates of photosynthetic induction, and these effects were explained by changes in diffusional and biochemical limitations. Maximising the rates of Rubisco activation would increase CO2 assimilation by 6-10%, while maximising the rates of stomatal opening would increase assimilation by at most 1-2%, at the same time negatively affecting intrinsic water use efficiency.

In Chapter 4 it is explored whether the effects of CO2 concentration on dynamic photosynthesis are similar across various irradiance environments. Gain and loss of photosynthetic induction in several low irradiance treatments, as well as sinusoidal changes in irradiance, were studied using tomato leaves. Elevated CO2 concentration (800 ppm) enhanced the rate of photosynthetic induction by 4-12% (compared to 400 ppm) and decreased the loss of photosynthetic induction by 21-25%. Elevated CO2 concentration enhanced rates of dynamic photosynthesis regardless of initial photosynthetic induction state to a similar extent. Therefore, rising global CO2 concentration will benefit integrated assimilation throughout whole canopies, where different leaf layers experience widely differing irradiance regimes.

In Chapter 5 it is tested whether stomatal limitation exists during photosynthetic induction in tomato leaves. The abscisic acid-deficient flacca mutant and its wildtype were exposed to various CO2 concentrations to change the diffusion gradient. Despite gs being much larger in flacca, photosynthetic induction proceeded with the same speed in both genotypes in ambient CO2 concentration. This suggested that stomata did not limit photosynthetic induction in the wildtype. Using these findings, several indices of stomatal limitations were compared. Diffusional limitation, a new index, was found to be the most useful.

In Chapter 6, an exploration of some physiological limitations underlying dynamic photosynthesis is undertaken. Several mutants, transformants and ecotypes of A. thaliana, affecting rates of Rubisco activation, gs, NPQ and sucrose metabolism, were used. Next to a characterisation of their steady-state responses to CO2 concentrations and irradiance, leaves were exposed to stepwise increases and decreases in irradiance (using several intensities) and to lightflecks of several amplitudes and frequencies. Rubisco activase isoform and concentration, as well as various levels of gs, strongly affected rates of dynamic photosynthesis, while this was not the case with low NPQ or sucrose phosphate synthase concentration. This suggests Rubisco activase and gs as targets for improvement of photosynthesis in fluctuating irradiance.

Chapter 7 is a modelling exercise of dynamic photosynthesis, based on data obtained from measurements on mutants of A. thaliana (Chapter 6). This includes a goal-seeking model that allows reproducing the regulation of Rubisco by irradiance and CO2 concentration. The model also includes a full description of leaf-level NPQ, incorporates mesophyll conductance and accounts for the fundamental physics of delays introduced by open gas exchange systems on CO2 measurements. Different data sets for model calibration and validation were used. It was found that the model accurately predicted the effects of the mutants, suggesting that the assumptions of the model were sound and represented the underlying mechanisms correctly.

In Chapter 8, the findings in this thesis are synthesized. The insights gained throughout this dissertation are related to existing literature to give a comprehensive overview of the state of knowledge about the limitations of dynamic photosynthesis. The methodology of assessing transient stomatal limitations, and some aspects of using chlorophyll fluorescence measurements during photosynthetic induction, are discussed. Finally, possible applications and ideas for future research on photosynthesis in fluctuating irradiance are discussed.

Genetics and regulation of combined abiotic and biotic stress tolerance in tomato
Kissoudis, C. - \ 2016
University. Promotor(en): Richard Visser, co-promotor(en): Gerard van der Linden; Yuling Bai. - Wageningen : Wageningen University - ISBN 9789462576568 - 212 p.
solanum lycopersicum - tomatoes - disease resistance - stress tolerance - defence mechanisms - plant diseases - abiotic injuries - stress response - phenotypic variation - genetic analysis - plant breeding - salt tolerance - tomaten - ziekteresistentie - stresstolerantie - verdedigingsmechanismen - plantenziekten - abiotische beschadigingen - stressreactie - fenotypische variatie - genetische analyse - plantenveredeling - zouttolerantie

Projections on the impact of climate change on agricultural productivity foresee prolonged and/or increased stress intensities and enlargement of a significant number of pathogens habitats. This significantly raises the occurrence probability of (new) abiotic and biotic stress combinations. With stress tolerance research being mostly focused on responses to individual stresses, our understanding of plants’ ability to adapt to combined stresses is limited.

In an attempt to bridge this knowledge gap, we hierarchized in chapter 1 existing information on individual abiotic or biotic stress adaptation mechanisms taking into consideration different anatomical, physiological and molecular layers of plant stress tolerance and defense. We identified potentially crucial regulatory intersections between abiotic and biotic stress signalling pathways following the pathogenesis timeline, and emphasized the importance of subcellular to whole plant level interactions by successfully dissecting the phenotypic response to combined stress. We considered both explicit and shared adaptive responses to abiotic and biotic stress, which included amongst others R-gene and systemic acquired resistance as well as reactive oxygen species (ROS), redox and hormone signalling, and proposed breeding targets and strategies.

In chapter 3 we focused on salt stress and powdery mildew combination in tomato, a vegetable crop with a wealth of genetic resources, and started with a genetic study. S. habrochaites LYC4 was found to exhibit resistance to both salt stress and powdery mildew. A LYC4 introgression line (IL) population segregated for both salt stress tolerance and powdery mildew resistance. Introgressions contributing to salt tolerance, including Na+ and Cl- accumulation, and powdery mildew resistance were precisely pinpointed with the aid of SNP marker genotyping. Salt stress (100mM NaCl) combined with powdery mildew infection increased the susceptibility of the population to powdery mildew in an additive manner, while decreasing the phenotypic variation for this trait. Only a few overlapping QTLs for disease resistance and salt stress tolerance were identified (one on a short region at the top of Chromosome 9 where numerous receptor-like kinases reside). Most genetic loci were specific for either salt stress tolerance or powdery mildew resistance indicating distinct genetic architectures. This enables genetic pyramiding approaches to build up combined stress tolerance.

Considering that abiotic stress in nature can be of variable intensities, we evaluated selected ILs under combined stress with salinity ranging from mild to severe (50, 100 and 150mM NaCl) in chapter 4. Mild salt stress (50mM) increased powdery mildew susceptibility and was accompanied by accelerated cell death-like senescence. On the contrary, severe salt stress (150mM) reduced the disease symptoms and this correlated with leaf Na+ and Cl- content in the leaves. The effects of salt stress on powdery mildew resistance may be dependent on resistance type and mechanisms. Near Isogenic Lines (NILs) that carry different PM resistance genes (Ol-1 (associated with slow hypersensitivity response, HR), ol-2 (an mlo mutant associated with papilla formation) and Ol-4 (an R gene associated with fast HR) indeed exhibited differential responses to combined stress. NIL-Ol-1 resembled the LYC4 ILs response, while NIL-ol-2 and NIL-Ol-4 maintained robust resistance and exhibited no senescence symptoms across all combinations, despite the observed reduction in callose deposition in NIL-ol-2. Increased susceptibility, senescence and fitness cost of NIL-Ol-1 under combined stress coincided with high induction of ethylene and jasmonate biosynthesis and response pathways, highly induced expression of cell wall invertase LsLIN6, and a reduction in the expression of genes encoding for antioxidant enzymes. These observations underlined the significance of stress intensity and mechanism of resistance to the outcome of salt stress and powdery mildew combination, underscoring the involvement of ethylene signalling to the susceptibility response under combined stress.

To examine the significance of hormone signalling in combined stress responses we evaluated crosses of tomato hormone mutants notabilis (ABA-deficient), defenseless1 (JA-deficient) and epinastic (ET overproducer) with NIL-Ol-1, NIL-ol-2 and NIL-Ol-4 in chapter 5. The highly pleiotropic epinastic mutant increased susceptibility of NIL-Ol-1, but decreased the senescence response under combined stress, and resulted in partial breakdown of NIL-ol-2 resistance, accompanied by reduced callose deposition. The effects of ET overproduction on susceptibility were more pronounced under combined stress. ABA deficiency in notabilis on the other hand greatly reduced susceptibility of NIL-Ol-1under combined stress at the expense of stronger growth reduction, and induced ROS overproduction. Partial resistance breakdown in the ol-2xnotabilis mutant accompanied by reduced callose deposition was observed, and this was restored under combined stress. Jasmonic acid deficiency phenotypic effects in defenseless mutants were subtle with modest increase in susceptibility for NIL-Ol-1 and NIL-ol-2. For NIL-ol-2 this increased susceptibility was reverted under combined stress. NIL-Ol-4 resistance remained robust across all mutant and treatment combinations. These results highlight the catalytic role of ET and ABA signalling on susceptibility and senescence under combined stress, accentuating concomitantly the importance of signalling fine tuning to minimize pleiotropic effects.

The potential of exploiting transcription factors to enhance tolerance to multiple stress factors and their combination was investigated in chapter 6 through the identification and functional characterization of tomato homologues of AtWRKYs 11, 29, 48, 70 and 72. Thirteen tomato WRKY homologues were identified, of which 9 were overexpressed (using transformation with A. tumefaciens) and 12 stably silenced via RNAi in tomato cultivar Money Maker (MM). SlWRKY11-OE and SlWRKY23-OE overexpressors and RNAi lines of SlWRKY7 and SlWRKY9 showed both increased biomass and relative salt tolerance. SlWRKY6-OE exhibited the highest relative salt stress tolerance, but had strongly decreased growth under control conditions. Exceptional phenotypes under control conditions were observed for SlWRKY10-OE (stunted growth) and SlWRKY23-RNAi (necrotic symptoms). These phenotypes were significantly restored under salt stress, and accompanied by decreased ROS production. Both lines exhibited increased resistance to powdery mildew, but this resistance was compromised under salt stress combination, indicating that these genes have important functions at the intersection of abiotic and biotic stress adaptation. SlWRKY23 appears to have a key regulatory role in the control of abiotic stress/defense and cell death control.

Experimental observations are critically discussed in the General Discussion with emphasis on potential distinctive responses in different pathosystems and abiotic and biotic stress resistance mechanisms as well as genetic manipulations for effectively achieving combined stress tolerance. This includes deployment of individual common regulators as well as pyramiding of non-(negatively) interacting components such as R-genes with abiotic stress resistance genes, and their translation potential for other abiotic and biotic stress combinations. Understanding and improving plant tolerance to stress combinations can greatly contribute to accelerating crop improvement towards sustained or even increased productivity under stress.

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