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.

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.

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.

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.

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.

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.

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.

MicroRNA534a control of BLADE-ON-PETIOLE 1 and 2 mediates juvenile-to-adult gametophyte transition in Physcomitrella patens.

The Arabidopsis thaliana BLADE-ON-PETIOLE genes encode a pair of transcriptional coactivators that regulate lateral organ architecture by promoting cell differentiation in their proximal regions. To gain insight into the roles of BOP genes early in land plant evolution, we characterized the functions of Physcomitrella patens BOP1 and BOP2 and their negative regulator Pp-miR534a. We show that in ΔPpMIR534a mutants lacking mature Pp-miR534a, cleavage of PpBOP1/2 is abolished, leading to elevated PpBOP1/2 transcript levels. These loss-of-function mutants display an accelerated gametophore development thus correlating elevated levels of PpBOP1/2 with premature bud formation. This is further supported by our finding that exposure to cytokinin, which is known to induce bud formation on caulonema, downregulates PpMIR534a transcription and increases the accumulation of PpBOP1 in apical caulonema cells. Reporter gene fusions showed that PpMIR534a is ubiquitously expressed in protonema whereas PpBOP1/2 accumulation is restricted almost exclusively to potent caulonema apical cells and their side branch initials, but absent from differentiated cells. Together, our data propose that PpBOP1/2 act as positive regulators of protonema differentiation and that Pp-miR534a is required to control the timing of the juvenile-to-adult gametophyte transition by spatially restricting their expression to caulonema stem cells. As protonemata develop, increased cytokinin levels downregulate Pp-MIR534a transcription in these cells until a threshold level of PpBOP1/2 is reached that triggers cell differentiation and bud formation.

Development of a transgenic early flowering pear (Pyrus communis L.) genotype by RNAi silencing of PcTFL1-1 and PcTFL1-2.

Trees require a long maturation period, known as juvenile phase, before they can reproduce, complicating their genetic improvement as compared to annual plants. 'Spadona', one of the most important European pear (Pyrus communis L.) cultivars grown in Israel, has a very long juvenile period, up to 14 years, making breeding programs extremely slow. Progress in understanding the molecular basis of the transition to flowering has revealed genes that accelerate reproductive development when ectopically expressed in transgenic plants. A transgenic line of 'Spadona', named Early Flowering-Spadona (EF-Spa), was produced using a MdTFL1 RNAi cassette targeting the native pear genes PcTFL1-1 and PcTFL1-2. The transgenic line had three T-DNA insertions, one assigned to chromosome 2 and two to chromosome 14 PcTFL1-1 and PcTFL1-2 were completely silenced, and EF-Spa displayed an early flowering phenotype: flowers developed already in tissue culture and on most rooted plants 1-8 months after transfer to the greenhouse. EF-Spa developed solitary flowers from apical or lateral buds, reducing vegetative growth vigor. Pollination of EF-Spa trees generated normal-shaped fruits with viable F1 seeds. The greenhouse-grown transgenic F1 seedlings formed shoots and produced flowers 1-33 months after germination. Sequence analyses, of the non-transgenic F1 seedlings, demonstrated that this approach can be used to recover seedlings that have no trace of the T-DNA. Thus, the early flowering transgenic line EF-Spa obtained by PcTFL1 silencing provides an interesting tool to accelerate pear breeding.

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.

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.

Flower development in the passion fruit Passiflora edulis requires a photoperiod-induced systemic graft-transmissible signal.

Different organisms use gradual seasonal changes in photoperiod to correctly time diverse developmental processes, such as transition to flowering in plants. Florigen is a systemic signal formed in leaves exposed to specific environmental cues, mainly photoperiodic, and capable of triggering flower induction in several species. Here we show that in Passiflora edulis, a perennial climbing vine, flower initiation occurs throughout the year; however, without long photoperiods, flower primordia show arrested growth and differentiation at an early stage. Our results support the existence of a positive, systemic, graft-transmissible signal, produced in mature leaves under LDs, that is required for normal flower development beyond sepal formation. Our results also suggest that Gibberellin acts to inhibit flower development. We provide evidence for genetic variation in the response to short photoperiods. A genotype capable of forming developed flowers under short photoperiods produces a positive graft transmissible signal allowing normal flower development under short days in a cultivar which normally aborts flower development under these conditions. We believe these findings contribute towards discovering the chemical nature of this interesting mobile signal involved in flower development.

Overexpression of the CBF2 transcriptional activator in Arabidopsis delays leaf senescence and extends plant longevity.

Leaf senescence is a programmed developmental process governed by various endogenous and exogenous factors, such as the plant developmental stage, leaf age, phytohormone levels, darkness, and exposure to stresses. It was found that, in addition to its well-documented role in the enhancement of plant frost tolerance, overexpression of the C-repeat/dehydration responsive element binding factor 2 (CBF2) gene in Arabidopsis delayed the onset of leaf senescence and extended the life span of the plants by approximately 2 weeks. This phenomenon was exhibited both during developmental leaf senescence and during senescence of detached leaves artificially induced by either darkness or phytohormones. Transcriptome analysis using the Affymetrix ATH1 genome array revealed that overexpression of CBF2 significantly influenced the expression of 286 genes in mature leaf tissue. In addition to 30 stress-related genes, overexpression of CBF2 also affected the expression of 24 transcription factor (TF) genes, and 20 genes involved in protein metabolism, degradation, and post-translational modification. These results indicate that overexpression of CBF2 not only increases frost tolerance, but also affects other developmental processes, most likely through interactions with additional TFs and protein modification genes. The present findings shed new light on the crucial relationship between plant stress tolerance and longevity, as reported for other eukaryotic organisms.

Arabidopsis KNOXI proteins activate cytokinin biosynthesis.

Plant architecture is shaped through the continuous formation of organs by meristems. Class I KNOTTED1-like homeobox (KNOXI) genes are expressed in the shoot apical meristem (SAM) and are required for SAM maintenance. KNOXI proteins and cytokinin, a plant hormone intimately associated with the regulation of cell division, share overlapping roles, such as meristem maintenance and repression of senescence, but their mechanistic and hierarchical relationship have yet to be defined. Here, we show that activation of three different KNOXI proteins using an inducible system resulted in a rapid increase in mRNA levels of the cytokinin biosynthesis gene isopentenyl transferase 7 (AtIPT7) and in the activation of ARR5, a cytokinin response factor. We further demonstrate a rapid and dramatic increase in cytokinin levels following activation of the KNOXI protein SHOOT MERISTEMLESS (STM). Application of exogenous cytokinin or expression of a cytokinin biosynthesis gene through the STM promoter partially rescued the stm mutant. We conclude that activation of cytokinin biosynthesis mediates KNOXI function in meristem maintenance. KNOXI proteins emerge as central regulators of hormone levels in plant meristems.

Ambient temperature perception in plants.

Since plants are sessile they must be able to sense and rapidly respond to changes in ambient temperature. Key aspects of plant development, including the transition to flowering and the circadian clock, have important inputs from ambient temperature. In the model system Arabidopsis thaliana, molecular candidates for mediating these roles have recently been uncovered, which will be critical for obtaining an understanding of the mechanisms involved.

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).

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.

Isolation of a dehydrin cDNA from orange and grapefruit citrus fruit that is specifically induced by the combination of heat followed by chilling temperatures.

Dehydrins (DHNs; late embryogenesis abundant D-11) are a family of plant proteins induced in response to environmental stresses such as water stress, salinity and freezing or which occur during the late stages of embryogenesis. Previously, it was reported that citrus contains a small gene family encoding a unique class of dehydrins that differs from most other plant dehydrins in various respects, such as having an unusual K-segment similar to that of gymnosperms. In the present study, we identified by cDNA differential display analysis a 'Navel' orange 202-bp polymerase chain reaction (PCR) fragment, which encoded the typical plant angiosperm-type K-segment consensus sequence, and of which the expression was down-regulated by exposure to low oxygen levels. The full-length cDNA sequence of the orange DHN, designated csDHN (for Citrus sinensis DHN), was further isolated by 5'-and 3'-RACE; it had a total length of 933 bp and encoded a predicted polypeptide of 235 amino acids. In addition, the same 202-bp 'Navel' dehydrin PCR fragment was used to screen a 'Star Ruby' grapefruit flavedo cDNA library, and its full-length grapefruit homologue, designated cpDHN (for C. paradisi DHN) was isolated and found to have a total length of 1024 bp and to encode a predicted polypeptide of 234 amino acids. The defined orange and grapefruit DHN proteins were completely identical in the 196 amino acids of their N-terminus but differed in their C-terminus region. Overall, the csDHN and cpDHN proteins share 84% identity and contain the conserved dehydrin serine cluster (S-segment) and a putative nuclear localization signal, but csDHN has one conserved dehydrin K-segment consensus sequence, whereas cpDHN contains two dehydrin K-segments. Both csDHN and cpDHN represent single copy genes, in 'Navel' orange and 'Star Ruby' grapefruit genomes, respectively. We found that the cpDHN gene was consistently expressed in the fruit peel tissue at harvest, but that its message levels dramatically decreased during storage at either ambient or low temperatures. However, a pre-storage hot water treatment, given to enhance fruit-chilling tolerance, increased cpDHN mRNA levels during the first 3 weeks of cold storage at 2 degrees C, and enabled the message levels to be retained for up to a further 8 weeks of cold storage at 2 degrees C. The hot water treatment by itself had no inductive effect on cpDHN gene expression when the fruits were held at non-chilling temperatures. Other stresses applied to the fruit, such as wounding, UV irradiation, water stress, low oxygen and exposure to the stress hormone ethylene decreased DHN mRNA levels, whereas abscisic acid had no effect at all.