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

Expression analysis of Flowering related genes in olive plants transformed with the "Medicago truncatula" FT gene MtFTa1

Olive tree (Olea europaea L.) forms inflorescences in lateral buds that flower in spring. Flowering occurs due to the presence of a mobile flower-promoting factor called florigen, the product of “FLOWERING LOCUS T” (FT). In many plants, FT and TERMINAL FLOWER 1 (TFL1) genes encode related proteins with opposite functions, i.e. FT induces flowering, while TFL1 represses it. Olive flower induction seems to be mediated by an increase in FT levels in response to cold winters. Because of climate change, warmer winters are expected, which can alter flowering time. Three olive transgenic lines containing the MtFTa1 gene from Medicago truncatula were obtained (FT5, FT7 and FT15) to study the effect of FT on flowering time (Haberman et al., 2017). The embryogenic line P1 from a seed of cv. Picual was used for transformation, and also as control (CP1). FT7 flowered continuously; FT5 did not flower and showed a dwarf branching phenotype, and FT15 had a dwarf-branching habit and developed abnormal flowers. The expression of the transgene and three endogenous genes (OeFT1, OeFT2 and OeTFL1-1) was analyzed in these juvenile plants, as well as in the control (CP1), throughout the year (autumn, winter and spring).Proyecto de Excelencia P11-AGR-7992, Junta de Andalucí

Transformación genética de olivo (Olea europaea L.) con un gen de Medicago truncatula que codifica para un proteína tipo FT

En el olivo (Olea europaea L.), la modificación de la arquitectura de la planta y la reducción del periodo juvenil son caracteres de interés en la mejora. Las proteínas codificadas por genes tipo FT (FLOWERING LOCUS T), además de actuar como componente principal de la señal sistémica inductora de floración conocida como florígeno, participan en la regulación de otros procesos del desarrollo en plantas, entre ellos, la determinación de la arquitectura o la dormancia. El objetivo de este trabajo es abordar la transformación genética de olivo con el gen MtFTa1 de Medicago truncatula, para estudiar su aplicación en la mejora. La transformación genética se llevó a cabo utilizando embriones somáticos, derivados de radícula de embrión zigótico, siguiendo el protocolo previamente establecido en nuestro laboratorio (Torreblanca et al. 2010, PCTOC 103:61-69). Se utilizaron la cepa de Agrobacterium AGL-1 y el vector binario Pro35S:MtFTA portando el gen nptII como gen de selección y el gen MtFTa1 bajo el control del promotor constitutivo CaMV35S. La regeneración de plantas se realizó siguiendo el protocolo previamente desarrollado en nuestro grupo de trabajo (Cerezo et al. 2011, PCTOC 106:337-344). Se obtuvo una tasa de transformación del 2,5%, recuperándose quince líneas transgénicas independientes. La expresión del transgén se analizó mediante qRT-PCR. En tres de las seis líneas con mayores niveles de expresión se observó floración precoz in vitro, mientras que las otras tres líneas transgénicas florecieron en invernadero de confinamiento, transcurridos 18-36 meses desde su aclimatación. Las flores obtenidas presentaron morfología irregular y no produjeron polen viable. Además, las plantas mostraron alteraciones en el crecimiento, como pérdida de dominancia apical y desarrollo continuo de yemas laterales. Por otro lado, en plantas aclimatadas que no florecieron tan precozmente, también se observó un mayor grado de ramificación en el eje principal en relación a las plantas control, con una menor longitud de entrenudos y mayor porcentaje de yemas axilares brotadas en ramos laterales de primer orden. Los resultados de este trabajo ponen de manifiesto el papel del gen FT en la regulación de la floración y arquitectura de las plantas de olivo.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Proyecto de Excelencia Junta de Andalucía P11-AGR-7992

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

FKF1 conveys timing information for CONSTANS stabilization in photoperiodic flowering

Plants use day-length information to coordinate flowering time with the appropriate season to maximize reproduction. In Arabidopsis, the long-day specific expression of CONSTANS (CO) protein is crucial for flowering induction. Although light signaling regulates CO protein stability, the mechanism by which CO is stabilized in the long-day afternoon has remained elusive. Here we demonstrate that FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) protein stabilizes CO protein in the afternoon in long days. FKF1 interacts with CO through its LOV domain, and blue light enhances this interaction. In addition, FKF1 simultaneously removes CYCLING DOF FACTOR 1 (CDF1) that represses CO and FLOWERING LOCUS T (FT) transcription. Together with CO transcriptional regulation, FKF1 protein controls robust FT mRNA induction through multiple feedforward mechanisms that accurately control flowering timing

The Flowering Integrator FT Regulates SEPALLATA3 and FRUITFULL Accumulation in Arabidopsis Leaves

The transition to flowering involves major changes in the shoot apical meristem and in the fate of existing leaf primordia. Transcripts of the Arabidopsis thaliana flowering-promoting gene FLOWERING LOCUS T (FT) are present in leaf tissue but can also promote flowering when artificially introduced into the meristem. FT may normally act in the leaf and/or the meristem, initiating or constituting a mobile flower-promoting signal. We studied FT-dependent events in the rosette leaf, some of which might precede or mimic events in the meristem and its primordia. We show FT-dependent transcript accumulation of the MADS box transcription factors FRUITFULL (FUL) and SEPALLATA3 (SEP3) in leaves. Abnormally high levels of FT further increase the expression of these genes, leading to morphological changes in the leaves. Loss of the flowering-time gene FD, as well as environmental conditions that delay flowering, reduce FT's effect on leaves via reduced activation of its targets. FUL, SEP3, and APETALA1 accumulation in the meristem is associated with and contributes to the transition to flowering. We propose that FT functions through partner-dependent transcriptional activation of these and as-yet-unknown genes and that this occurs at several sites. Organ fate may depend on both degree of activation and the developmental stage reached by the organ before activation occurs

Isolation of four heat shock protein cDNAs from grapefruit peel tissue and characterization of their expression in response to heat and chilling temperature stresses

In order to continuously supply horticultural products for long periods, it is essential to store them after harvest in low temperatures. However, many tropical and subtropical fruits and vegetables, such as citrus, are sensitive to chilling. In previous studies, the authors have shown that a short hot water rinsing treatment (at 62degreesC for 20 s) increased chilling tolerance in grapefruit. In order to gain more insight into the molecular mechanisms involved in heat-induced chilling tolerance, PCR cDNA subtraction analysis was performed which isolated four different PCR fragments whose expression was enhanced 24 h after the heat treatment, and that showed high sequence homology with various plant HSP18-I, HSP18-II, HSP22 and HSP70 genes. It was found that the short hot water treatment given at 62degreesC for 20 s, but not at lower temperatures of 20 or 53degreesC, increased the expression of the various HSP cDNAs in grapefruit peel tissue. However, when the fruits were kept at ambient temperatures, the increases in HSP mRNA levels following the hot water treatment were temporary and lasted only between 6 and 48 h. Similar temporary increases in the HSP mRNA levels were detected following exposure of the fruit to a hot air treatment at 40degreesC for 2 h. Nevertheless, when the fruits were treated with hot water but afterwards stored at chilling temperatures of 2degreesC, the mRNA levels of the various HSP18-I, HSP18-II, HSP22 and HSP70 cDNAs increased and remained high and stable during the entire 8-week cold-storage period, suggesting their possible involvement in heat-induced chilling-tolerance responses. The chilling treatment by itself increased the expression of the HSP18-I cDNA, but had no effect on the mRNA levels of any of the other HSP cDNAs. Exposure of fruit to other stresses, such as wounding, UV irradiation, anaerobic conditions and exposure to ethylene, had no effect on the expression of the various HSPs. Overall, the study explored the correlation between the expression and persistence of various HSP cDNAs in grapefruit peel tissue during cold storage, on the one hand, and the acquisition of chilling tolerance, on the other hand, and the results suggest that HSPs may play a general role in protecting plant cells under both high- and low-temperature stresses