Effect of heterologous expression of FT gene from Medicago truncatula in growth and flowering behavior of olive plants.

Olive (Olea europaea L. subsp. europaea) is one of the most important crops of the Mediterranean Basin and temperate areas worldwide. Obtaining new olive varieties adapted to climatic changing conditions and to modern agricultural practices, as well as other traits such as biotic and abiotic stress resistance and increased oil quality, is currently required; however, the long juvenile phase, as in most woody plants, is the bottleneck in olive breeding programs. Overexpression of genes encoding the 'florigen' Flowering Locus T (FT), can cause the loss of the juvenile phase in many perennials including olives. In this investigation, further characterization of three transgenic olive lines containing an FT encoding gene from Medicago truncatula, MtFTa1, under the 35S CaMV promoter, was carried out. While all three lines flowered under in vitro conditions, one of the lines stopped flowering after acclimatisation. In soil, all three lines exhibited a modified plant architecture; e.g., a continuous branching behaviour and a dwarfing growth habit. Gene expression and hormone content in shoot tips, containing the meristems from which this phenotype emerged, were examined. Higher levels of OeTFL1, a gene encoding the flowering repressor TERMINAL FLOWER 1, correlated with lack of flowering. The branching phenotype correlated with higher content of salicylic acid, indole-3-acetic acid and isopentenyl adenosine, and lower content of abscisic acid. The results obtained confirm that heterologous expression of MtFTa1 in olive induced continuous flowering independently of environmental factors, but also modified plant architecture. These phenotypical changes could be related to the altered hormonal content in transgenic plants.

Studying Parameters Affecting Accumulation of Chilling Units Required for Olive Winter Flower Induction.

With global warming, mean winter temperatures are predicted to increase. Therefore, understanding how warmer winters will affect the levels of olive flower induction is essential for predicting the future sustainability of olive oil production under different climactic scenarios. Here, we studied the effect of fruit load, forced drought in winter, and different winter temperature regimes on olive flower induction using several cultivars. We show the necessity of studying trees with no previous fruit load as well as provide evidence that soil water content during winter does not significantly affect the expression of an FT-encoding gene in leaves and the subsequent rate of flower induction. We collected yearly flowering data for 5 cultivars for 9 to 11 winters, altogether 48 data sets. Analyzing hourly temperatures from these winters, we made initial attempts to provide an efficient method to calculate accumulated chill units that are then correlated with the level of flower induction in olives. While the new models tested here appear to predict the positive contribution of cold temperatures, they lack in accurately predicting the reduction in cold units caused by warm temperatures occurring during winter.

Expression Profiling of Four Mango FT/TFL1-Encoding Genes under Different Fruit Load Conditions, and Their Involvement in Flowering Regulation.

Plant flowering is antagonistically modulated by similar FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1) proteins. In mango (Mangifera indica L.), flowering is induced by cold temperatures, unless the tree is juvenile or the adult tree had a high fruit load (HFL) in the summer. Here, we studied the effects of juvenility and fruit load on the expression of four MiFT/TFL1 genes cloned from the mango 'Shelly' cultivar. Ectopic expression of MiFT1 in Arabidopsis resulted in early flowering, whereas over-expression of MiFT2 and the two cloned MiTFL1 genes repressed flowering. Moreover, juvenility was positively correlated with higher transcript levels of MiFT2 and both MiTFL1s. In trees with a low fruit load, leaf MiFT1 expression increased in winter, whereas HFL delayed its upregulation. MiFT2 expression was upregulated in both leaves and buds under both fruit load conditions. Downregulation of both MITFL1s in buds was associated with a decrease in regional temperatures under both conditions; nevertheless, HFL delayed the decrease in their accumulation. Our results suggest that cold temperature has opposite effects on the expression of MiFT1 and the MiTFL1s, thereby inducing flowering, whereas HFL represses flowering by both suppressing MiFT1 upregulation and delaying MiTFL1s downregulation. The apparent flowering-inhibitory functions of MiFT2 are discussed.

Determining Reproductive Parameters, which Contribute to Variation in Yield of Olive Trees from Different Cultivars, Irrigation Regimes, Age and Location.

Olive (Olea europaea L.) trees can reach a very old age and still bear fruit. Although traditional groves are planted at low density and are rainfed, many newer groves are planted at higher densities and irrigated. As expected, initial yields per area are larger in high density plantations, yet some farmers claim they experience a reduction in productivity with grove age, even in well maintained trees. In order to test the accuracy of this claim and its underlying cause, we measured several productivity parameters in selected branches of trees in seven sites differing in cultivar ('Barnea' or 'Souri'), location and irrigation regime (rainfed or irrigated) for two consecutive years. For each site (cultivar/location/regime), we compared neighboring groves of different ages, altogether 14 groves. There was no consistent reduction in productivity in older groves. Differences in productivity between irrigated cultivars were mostly due to variation in the percentage of inflorescences that formed fruit. Several parameters were higher in irrigated, compared to rainfed 'Souri'. Differences in productivity between years within the same grove was mostly due to variation in the percentage of nodes forming inflorescences. We studied the expression of OeFT2 encoding a FLOWERING LOCUS T protein involved in olive flower induction in leaves of trees of different ages, including juvenile seedlings. Expression increased during winter in mature trees and correlated with the percentage of inflorescences formed. The leaves of juvenile seedlings expressed higher levels of two genes encoding APETALA2-like proteins, potential inhibitors of OeFT2 expression. The buds of juvenile seedlings expressed higher levels of OeTFL1, encoding a TERMINAL FLOWER 1 protein, a potential inhibitor of OeFT2 function in the meristem. Our results suggest that olives, once past the juvenile phase, can retain a similar level of productivity even in densely planted well maintained groves.

Complete Genome Sequence of an Isolate of Passiflora chlorosis virus from Passion Fruit (Passiflora edulis Sims).

We report the first complete genome sequence of an isolate of Passiflora chlorosis virus (PaCV), a member of the Potyviridae family, identified in passion fruit (Passiflora edulis Sims) plants grown in Israel. The assembled genome is 9672 nucleotides long and encodes a 3084 amino acids polyprotein that is predicted to be proteolytically cleaved into 10 mature peptides. Our analysis of the genome sequence shows that PaCV is a distinct species, sharing 68.5% nucleotide sequence identity and 71.5% amino acid sequence identity with isolates of the bean common mosaic necrosis virus (BCMNV), the most closely related virus classified within the genus Potyvirus. Using quantitative PCR, we detected the virus in RNA samples from leaves exhibiting symptoms of infection, with higher levels in clearly chlorotic leaves, but not in those from healthy leaves.

Rapid starch degradation in the wood of olive trees under heat and drought is permitted by three stress-specific beta amylases.

Carbon reserve use is a major drought response in trees, enabling tree survival in conditions prohibiting photosynthesis. However, regulation of starch metabolism under drought at the whole-tree scale is still poorly understood. To this end, we combined measurements of nonstructural carbohydrates (NSCs), tree physiology and gene expression. The experiment was conducted outside on olive trees in pots under 90 d of seasonal spring to summer warming. Half of the trees were also subjected to limited water conditions for 28 d. Photosynthesis decreased in dehydrating trees from 19 to 0.5 µmol m-2  s-1 during the drought period. Starch degradation and mannitol production were a major drought response, with mannitol increasing to 71% and 41% out of total NSCs in shoots and roots, respectively. We identified the gene family members potentially relevant either to long-term or stress-induced carbon storage. Partitioning of expression patterns among ß amylase and starch synthase family members was observed, with three ß amylases possibly facilitating the rapid starch degradation under heat and drought. Our results suggest a group of stress-related, starch metabolism genes, correlated with NSC fluctuations during drought and recovery. The daily starch metabolism gene expression was different from the stress-mode starch metabolism pattern, where some genes are uniquely expressed during the stress-mode response.

Complete genome sequence of a novel virus, classifiable within the Potyviridae family, which infects passion fruit (Passiflora edulis).

We report the complete nucleotide sequence of a new member of the Potyviridae family isolated from passion fruit plants grown in Israel, called Passiflora edulis symptomless virus (PeSV). The PeSV genome is 9,928 nucleotides long and encodes a 3,173 amino acids polyprotein that is predicted to be proteolytically cleaved into 10 mature peptides. Our phylogenetic analysis shows that PeSV represents a new species, and is most closely related to rose yellow mosaic virus (RoYMV). According to currently accepted criteria for genus demarcation, both viruses should be assigned as representative isolates of new species in the recently approved genus, Roymovirus, in the Potyviridae family.

A possible role for flowering locus T-encoding genes in interpreting environmental and internal cues affecting olive (Olea europaea L.) flower induction.

Olive (Olea europaea L.) inflorescences, formed in lateral buds, flower in spring. However, there is some debate regarding time of flower induction and inflorescence initiation. Olive juvenility and seasonality of flowering were altered by overexpressing genes encoding flowering locus T (FT). OeFT1 and OeFT2 caused early flowering under short days when expressed in Arabidopsis. Expression of OeFT1/2 in olive leaves and OeFT2 in buds increased in winter, while initiation of inflorescences occurred i n late winter. Trees exposed to an artificial warm winter expressed low levels of OeFT1/2 in leaves and did not flower. Olive flower induction thus seems to be mediated by an increase in FT levels in response to cold winters. Olive flowering is dependent on additional internal factors. It was severely reduced in trees that carried a heavy fruit load the previous season (harvested in November) and in trees without fruit to which cold temperatures were artificially applied in summer. Expression analysis suggested that these internal factors work either by reducing the increase in OeFT1/2 expression or through putative flowering repressors such as TFL1. With expected warmer winters, future consumption of olive oil, as part of a healthy Mediterranean diet, should benefit from better understanding these factors.

Scanning Electron Microscope (SEM) Imaging to Determine Inflorescence Initiation and Development in Olive.

Here we present a protocol that describes how to image the structure of the olive axillary bud meristem with a scanning electron microscope (SEM) in order to characterize its identity and developmental stage. Briefly, the specimen is fixed with glutaraldehyde, saturated with ethanol, dried in a critical point dryer (CPD) system, dissected, coated with a conducting material and imaged with a scanning electron microscopy (SEM).

Different flowering response to various fruit loads in apple cultivars correlates with degree of transcript reaccumulation of a TFL1-encoding gene.

In many perennial fruit trees, flowering in the year following a year with heavy fruit load can be quite limited. This biennial cycle of fruiting, termed alternate bearing, was described 170 years ago in apple (Malus domestica). Apple inflorescences are mainly found on short branches (spurs). Bourse shoots (BS) develop from the leaf axils of the spur. BS apices may terminate ~100 days after flowering, with formation of next year's inflorescences. We sought to determine how developing fruit on the spur prevents the adjacent BS apex from forming an inflorescence. The presence of adjacent fruit correlated with reaccumulation of transcript encoding a potential flowering inhibitor, MdTFL1-2, in BS apices prior to inflorescence initiation. BS apices without adjacent fruit that did not flower due to late fruitlet removal, neighbouring fruit on the tree, or leaf removal, also reaccumulated the MdTFL1-2 transcript. Fruit load and gibberellin (GA) application had similar effects on the expression of MdTFL1-2 and genes involved in GA biosynthesis and metabolism. Some apple cultivars are less prone to alternate bearing. We show that the response of a BS apex to different numbers of adjacent fruit differs among cultivars in both MdTFL1-2 accumulation and return flowering. These results provide a working model for the further study of alternate bearing, and help clarify the need for cultivar-specific approaches to reach stable fruit production.

Expression of flowering locus T2 transgene from Pyrus communis L. delays dormancy and leaf senescence in Malus×domestica Borkh, and causes early flowering in tobacco.

Annual and perennial plants represent two different evolutionary strategies based on differential synchronization of their reproductive development. The mobile signal protein FLOWERING LOCUS T (FT) plays a central role in mediating the onset of reproduction in both plant types. Two novel FT-like genes from pear (Pyrus communis)-PcFT1 and PcFT2-were isolated, and their expression profiles were determined for one annual cycle. The effects of PcFT2 on flowering were investigated in annual (tobacco) and perennial (apple) plants by means of grafting and generating transgenic plants. Long-distance graft transmission of PcFT2 in both annual and perennial plants was confirmed using a 35S::PcFT2-YFP construct. Ectopic overexpression of PcFT2 caused early flowering in tobacco but not in apple. Transgenic apples were less sensitive to short-day-induced dormancy, and this phenotype was also observed in wild-type apples grafted onto the transgenic plants. Comparison of PcFT2 protein structure to the paralogous FT proteins from apple and pear showed alterations that could influence protein structure and thus the florigen-activation complex. PcFT2 protein seems to function by promoting flowering as all other FT proteins in the annual plant tobacco while in the perennial plant apple PcFT2 does not promote flowering but delays senescence. This observation may hint to a modified function of FT2 in perennial plants.

Expression profiling of FLOWERING LOCUS T-like gene in alternate bearing 'Hass' avocado trees suggests a role for PaFT in avocado flower induction.

In many perennials, heavy fruit load on a shoot decreases the ability of the plant to undergo floral induction in the following spring, resulting in a pattern of crop production known as alternate bearing. Here, we studied the effects of fruit load on floral determination in 'Hass' avocado (Persea americana). De-fruiting experiments initially confirmed the negative effects of fruit load on return to flowering. Next, we isolated a FLOWERING LOCUS T-like gene, PaFT, hypothesized to act as a phloem-mobile florigen signal and examined its expression profile in shoot tissues of on (fully loaded) and off (fruit-lacking) trees. Expression analyses revealed a strong peak in PaFT transcript levels in leaves of off trees from the end of October through November, followed by a return to starting levels. Moreover and concomitant with inflorescence development, only off buds displayed up-regulation of the floral identity transcripts PaAP1 and PaLFY, with significant variation being detected from October and November, respectively. Furthermore, a parallel microscopic study of off apical buds revealed the presence of secondary inflorescence axis structures that only appeared towards the end of November. Finally, ectopic expression of PaFT in Arabidopsis resulted in early flowering transition. Together, our data suggests a link between increased PaFT expression observed during late autumn and avocado flower induction. Furthermore, our results also imply that, as in the case of other crop trees, fruit-load might affect flowering by repressing the expression of PaFT in the leaves. Possible mechanism(s) by which fruit crop might repress PaFT expression, are discussed.

Genetic variation in yield under hot ambient temperatures spotlights a role for cytokinin in protection of developing floral primordia.

Unusually hot ambient temperatures (HAT) can cause pre-anthesis abortion of flowers in many diverse species, limiting crop production. This limitation is becoming more substantial with climate change. Flower primordia of passion fruit (Passiflora edulis Sims) vines exposed to HAT summers, normally abort. Flower abortion can also be triggered by gibberellin application. We screened for, and identified a genotype capable of reaching anthesis during summer as well as controlled HAT conditions, and also more resistant to gibberellin. Leaves of this genotype contained higher levels of endogenous cytokinin. We investigated a possible connection between higher cytokinin levels and response to gibberellin. Indeed, the effects of gibberellin application were partially suppressed in plants pretreated with cytokinin. Can higher cytokinin levels protect flowers from aborting under HAT conditions? In passion fruit, flowers at a specific stage showed more resistance in response to HAT after cytokinin application. We further tested this hypothesis in Arabidopsis. Transgenic lines with high or low cytokinin levels and cytokinin applications to wild-type plants supported a protective role for cytokinin on developing flowers exposed to HAT. Such findings may have important implications in future breeding programmes as well as field application of growth regulators.

Shielding Flowers Developing under Stress: Translating Theory to Field Application.

Developing reproductive organs within a flower are sensitive to environmental stress. A higher incidence of environmental stress during this stage of a crop plants' developmental cycle will lead to major breaches in food security. Clearly, we need to understand this sensitivity and try and overcome it, by agricultural practices and/or the breeding of more tolerant cultivars. Although passion fruit vines initiate flowers all year round, flower primordia abort during warm summers. This restricts the season of fruit production in regions with warm summers. Previously, using controlled chambers, stages in flower development that are sensitive to heat were identified. Based on genetic analysis and physiological experiments in controlled environments, gibberellin activity appeared to be a possible point of horticultural intervention. Here, we aimed to shield flowers of a commercial cultivar from end of summer conditions, thus allowing fruit production in new seasons. We conducted experiments over three years in different settings, and our findings consistently show that a single application of an inhibitor of gibberellin biosynthesis to vines in mid-August can cause precocious flowering of ~2-4 weeks, leading to earlier fruit production of ~1 month. In this case, knowledge obtained on phenology, environmental constraints and genetic variation, allowed us to reach a practical solution.

Isolation and characterization of FLOWERING LOCUS T subforms and APETALA1 of the subtropical fruit tree Dimocarpus longan.

Longan (Dimocarpus longan Lour.) is a subtropical evergreen fruit tree, mainly cultivated in Asia. Two putative floral integrator genes, D. longan FLOWERING LOCUS T1 and 2 (DlFT1 and DlFT2) were isolated and both translated sequences revealed a high homology to FT sequences from other plants. Moreover, two APETALA1-like (DlAP1-1 and DlAP1-2) sequences from longan were isolated and characterized. Results indicate that the sequences of these genes are highly conserved, suggesting functions in the longan flowering pathway. Ectopic expression of the longan genes in arabidopsis resulted in different flowering time phenotypes of transgenic plants. Expression experiments reveal a different action of the longan FT genes and indicate that DlFT1 is a flowering promoter, while DlFT2 acts as flowering inhibitor. Overexpression of longan AP1 genes in transgenic arabidopsis results in a range of flowering time phenotypes also including early and late flowering individuals.

Constraints to obtaining consistent annual yields in perennial tree crops. I: Heavy fruit load dominates over vegetative growth.

Farmers lack effective methods to achieve and maintain stable production from year to year in many commercial fruit crops. Annual fruit yield within a region often alternates between high and low fruit load and is termed alternate bearing. The underlying cause of alternate bearing is the negative impact of high fruit load on vegetative growth and next year's flowering. In this review, we emphasize common responses of diverse perennials to heavy crop load. We present botanical, ecological and horticultural perspectives on irregular bearing. The later part of this review focuses on understanding how high fruit load dominates over vegetative growth. We discuss sink strengths and putative mobile signals (hormones), perhaps seed-derived. We highlight gaps in current understanding of alternate bearing, and discuss new approaches to better understand fruit load dominance. Assuming the effect of high fruit load may be related to other mechanisms of sink partitioning, other forms of dominance are presented such as apical, first fruit and king fruit dominance. Dominance seems to be enforced, in independent cases through the establishment of a polar auxin transport system from the stronger sink. Once established this somehow perturbs the transport of auxin out of weaker sinks. Possibly, fruit derived auxin may alter the polar auxin transport system of the shoot to inhibit shoot growth.

Constraints to obtaining consistent annual yields in perennials. II: Environment and fruit load affect induction of flowering.

In many commercial fruit crop species, high fruit load inhibits vegetative growth and floral induction. As a result, trees that had a high fruit load will bear few flowers and fruit the following year, along with abundant vegetative growth. We previously discussed how high fruit load interferes with concurrent shoot growth. Here we focus on how high fruit load impacts the process of flowering. Ascertaining the precise time at which specific buds begin the floral transition in each species is challenging. The use of indirect approaches to determine time of floral induction or evocation may lead to questionable conclusions. Annual and perennial plants appear to use conserved proteins for flowering induction and initiation. The accumulation or reduction of transcripts encoding proteins similar to Arabidopsis (annual) FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1), respectively, correlates well with flower induction in several diverse species. The recent use of such markers provides a means to formulate an accurate timeframe for floral induction in different species and holds promise in providing new insight into this important developmental event. A role for hormones in modulating the inhibitory effect of fruit load on floral induction is also discussed.

Evolutionary, genetic, environmental and hormonal-induced plasticity in the fate of organs arising from axillary meristems in Passiflora spp.

Tendrils can be found in different plant species. In legumes such as pea, tendrils are modified leaves produced by the vegetative meristem but in the grape vine, a same meristem is used to either form a tendril or an inflorescence. Passiflora species originated in ecosystems in which there is dense vegetation and competition for light. Thus climbing on other plants in order to reach regions with higher light using tendrils is an adaptive advantage. In Passiflora species, after a juvenile phase, every leaf has a subtending vegetative meristem, and a separate meristem that forms both flowers and a tendril. Thus, flowers are formed once a tendril is formed yet whether or not this flower will reach bloom depends on the environment. For example, in Passiflora edulis flowers do not develop under shaded conditions, so that tendrils are needed to bring the plant to positions were flowers can develop. This separate meristem generally forms a single tendril in different Passiflora species yet the number and position of flowers formed from the same meristem diverges among species. Here we display the variation among species as well as variation within a single species, P. edulis. We also show that the number of flowers within a specific genotype can be modulated by applying Cytokinins. Finally, this separate meristem is capable of transforming into a leaf-producing meristem under specific environmental conditions. Thus, behind what appears to be a species-specific rigid program regarding the fate of this meristem, our study helps to reveal a plasticity normally restrained by genetic, hormonal and environmental constraints.

Congratulations, you have been carefully chosen to represent an important developmental regulator!

BACKGROUND: Studying a process in a new species often relies on focusing our attention to a candidate gene, encoding a protein similar to one with a known function. Not all the choices seem to be prudent. SCOPE: This Viewpoint includes an overview of issues that are encountered during research of candidate genes. Defining a match for a gene of interest, deciding whether variation in ESTs or RNAseq data for a certain transcript, represent more than one gene. The problem of incorrect annotation of genes due to incorrect in-silico splicing, is also mentioned. The author's humble opinion on how to deal with these issues is provided. CONCLUSIONS: The vast amount of new sequence data provides us with great possibilities for giant leaps in our understanding. Still, we cannot afford to skip over the tedious steps required to confirm that we are indeed studying the correct gene, and try to be sure that the complex expression pattern we observe is not a composite of several genes.

Ripening attributes of new passion fruit line featuring seasonal non-climacteric behavior.

The passion fruit hybrid cultivar 'Passion Dream' (PD) produces two cycles of fruiting per year, in the summer and winter. Self-hybridization of PD created various lines, including 'Ripens during Summer' (RS), which lacks the ability to abscise during winter, suggesting a seasonal nonclimacteric behavior. The two lines were characterized by several quality traits: PD produced high ethylene levels in both seasons; RS produced significantly less ethylene during summer and almost none during winter. The ratio of total soluble solids to titratable acidity (TSS/TA), and aroma volatiles production, as determined by solid-phase microextraction/gas chromatograph-mass spectrometer (SPME/GC-MS) techniques, and taste indices were highest in PD summer fruits and lowest in RS winter fruits. Peel color in PD was affected by environmental and storage temperatures, whereas RS fruits always showed a strong purple color. The present findings suggest that ethylene production levels in passion fruit greatly influence various ripening processes, including acid degradation, increased TSS/TA ratio, accumulation of aroma volatiles, and tastiness.