Long-distance Signaling In Plants Research Articles

Optimization of cassava (Manihot esculenta Crantz) grafting technique to enhance its adoption in cassava cultivation

Grafting techniques have been successfully adopted to improve resistance to biotic and abiotic stresses, increase yields, fruit quality and study systemic signaling in plants. This technique has not been fully explored in cassava and there is currently no standardized grafting method for this species published especially in Africa. This is the first report on cassava grafting protocol in Africa with valuable advantages including utilizing a cost-effective and environmentally friendly wooden healing chamber. In this study, we describe an optimized cleft grafting protocol for cassava utilizing a wooden healing chamber and outline the step-by-step procedure with optimum conditions to generate a high grafting success rate. Using a top wedge grafting technique with high reproducibility and success rates, we developed a straightforward and robust grafting protocol for cassava (M. esculenta) cultivars. Grafting success was recorded and this protocol produced a high grafting success of 90 % and its reproducibility makes it suitable for mass production thereby addressing the need for efficient cassava propagation. This grafting protocol requires less specialized equipment and expertise making it more accessible to farmers and researchers with limited resources to promote the use of grafting for cassava growth, yield improvement and advanced studies such as systemic long-distance signaling in plants.•Optimization of cleft grafting method obtains a high success grafting rate of cassava.•A wooden healing chamber provides a controlled environment for graft healing.•Promoting cassava grafting; a priority to produce new cultivars and explore breeding research prospects.

Phosphorus homeostasis: acquisition, sensing, and long-distance signaling in plants.

Phosphorus (P), an essential nutrient required by plants often becomes the limiting factor for plant growth and development. Plants employ various mechanisms to sense the continuously changing P content in the soil. Transcription factors, such as SHORT ROOT (SHR), AUXIN RESPONSE FACTOR19 (ARF19), and ETHYLENE-INSENSITIVE3 (EIN3) regulate the growth of primary roots, root hairs, and lateral roots under low P. Crop improvement strategies under low P depend either on improving P acquisition efficiency or increasing P utilization. The various phosphate transporters (PTs) are involved in the uptake and transport of P from the soil to various plant cellular organelles. A plethora of regulatory elements including transcription factors, microRNAs and several proteins play a critical role in the regulation of coordinated cellular P homeostasis. Among these, the well-established P starvation signaling pathway comprising of central transcriptional factor phosphate starvation response (PHR), microRNA399 (miR399) as a long-distance signal molecule, and PHOSPHATE 2 (PHO2), an E2 ubiquitin conjugase is crucial in the regulation of phosphorus starvation responsive genes. Under PHR control, several classes of PHTs, microRNAs, and proteins modulate root architecture, and metabolic processes to enable plants to adapt to low P. Even though sucrose and inositol phosphates are known to influence the phosphorus starvation response genes, the exact mechanism of regulation is still unclear. In this review, a basic understanding of P homeostasis under low P in plants and all the above aspects are discussed.

A voltage-dependent Ca2+ homeostat operates in the plant vacuolar membrane.

Cytosolic calcium signals are evoked by a large variety of biotic and abiotic stimuli and play an important role in cellular and long distance signalling in plants. While the function of the plasma membrane in cytosolic Ca2+ signalling has been intensively studied, the role of the vacuolar membrane remains elusive. A newly developed vacuolar voltage clamp technique was used in combination with live-cell imaging, to study the role of the vacuolar membrane in Ca2+ and pH homeostasis of bulging root hair cells of Arabidopsis. Depolarisation of the vacuolar membrane caused a rapid increase in the Ca2+ concentration and alkalised the cytosol, while hyperpolarisation led to the opposite responses. The relationship between the vacuolar membrane potential, the cytosolic pH and Ca2+ concentration suggests that a vacuolar H+ /Ca2+ exchange mechanism plays a central role in cytosolic Ca2+ homeostasis. Mathematical modelling further suggests that the voltage-dependent vacuolar Ca2+ homeostat could contribute to calcium signalling when coupled to a recently discovered K+ channel-dependent module for electrical excitability of the vacuolar membrane.

Micrografting device for testing systemic signaling in Arabidopsis.

Grafting techniques have been applied in studies of systemic, long-distance signaling in several model plants. Seedling grafting in Arabidopsis, known as micrografting, enables investigation of the molecular mechanisms of systemic signaling between shoots and roots. However, conventional micrografting requires a high level of skill, limiting its use. Thus, an easier user-friendly method is needed. Here, we developed a silicone microscaled device, the micrografting chip, to obviate the need for training and to generate less stressed and more uniformly grafted seedlings. The chip has tandemly arrayed units, each of which consists of a seed pocket for seed germination and a micro-path with pairs of pillars for hypocotyl holding. Grafting, including seed germination, micrografting manipulation and establishment of tissue reunion, is performed on the chip. Using the micrografting chip, we evaluated the effect of temperature and the carbon source on grafting, and showed that a temperature of 27°C and a sucrose concentration of 0.5% were optimal. We also used the chip to investigate the mechanism of systemic signaling of iron status using a quadruple nicotianamine synthase (nas) mutant. The constitutive iron-deficiency response in the nas mutant because of iron accumulation in shoots was significantly rescued by grafting of wild-type shoots or roots, suggesting that shoot- and root-ward translocation of nicotianamine-iron complexes and/or nicotianamine is essential for iron mobilization. Thus, our micrografting chip will promote studies of long-distance signaling in plants.

Identification of miRNA precursors in the phloem of Cucurbita maxima.

Plant development and responses to environmental cues largely depend on mobile signals including microRNAs (miRNAs) required for post-transcriptional silencing of specific genes. Short-range cell-to-cell transport of miRNA in developing tissues and organs is involved in transferring positional information essential for determining cell fate. Among other RNA species, miRNAs are found in the phloem sap. Long-distance transport of miRNA via the phloem takes a part in regulation of physiological responses to changing environmental conditions. As shown for regulation of inorganic phosphorus and sulfate homeostasis, mature miRNAs rather than miRNAs precursors are transported in the phloem as signaling molecules. Here, a bioinformatics analysis of transcriptomic data for Cucurbita maxima phloem exudate RNAs was carried out to elucidate whether miRNA precursors could also be present in the phloem. We demonstrated that the phloem transcriptome contained a subset of C. maxima pri-miRNAs that differed from a subset of pri-miRNA sequences abundant in a leaf transcriptome. Differential accumulation of pri-miRNA was confirmed by PCR analysis of C. maxima phloem exudate and leaf RNA samples. Therefore, the presented data indicate that a number of C. maxima pri-miRNAs are selectively recruited to the phloem translocation pathway. This conclusion was validated by inter-species grafting experiments, in which C. maxima pri-miR319a was found to be transported across the graft union via the phloem, confirming the presence of pri-miR319a in sieve elements and showing that phloem miRNA precursors could play a role in long-distance signaling in plants.

Rapid, long-distance signaling in plants

Plant Science A plant injured on one leaf by a nibbling insect can alert its other leaves to begin anticipatory defense responses. Working in the model plant Arabidopsis , Toyota et al. show that this systemic signal begins with the release of glutamate, which is perceived by glutamate receptor–like ion channels (see the Perspective by Muday and Brown-Harding). The ion channels then set off a cascade of changes in calcium ion concentration that propagate through the phloem vasculature and through intercellular channels called plasmodesmata. This glutamate-based long-distance signaling is rapid: Within minutes, an undamaged leaf can respond to the fate of a distant leaf. Science , this issue p. [1112][1]; see also p. [1068][2] [1]: /lookup/doi/10.1126/science.aat7744 [2]: /lookup/volpage/361/1068?iss=6407

The Use of Grafting to Study Systemic Signaling in Plants.

Grafting has long been an important technique in agriculture. Nowadays, grafting is a widely used technique also to study systemic long-distance signaling in plants. Plants respond to their surrounding environment, and at that time many aspects of their physiology are regulated systemically; these start from local input signals and are followed by the transmission of information to the rest of the plant. For example, soil nutrient conditions, light/photoperiod, and biotic and abiotic stresses affect plants heterogeneously, and plants perceive such information in specific plant tissues or organs. Such environmental cues are crucial determinants of plant growth and development, and plants drastically change their morphology and physiology to adapt to various events in their life. Hitherto, intensive studies have been conducted to understand systemic signaling in plants, and grafting techniques have permitted advances in this field. The breakthrough technique of micrografting in Arabidopsis thaliana was established in 2002 and led to the development of molecular genetic tools in this field. Thereafter, various phenomena of systemic signaling have been identified at the molecular level, including nutrient fixation, flowering, circadian clock and defense against pathogens. The significance of grafting is that it can clarify the transmission of the stimulus and molecules. At present, many micro- and macromolecules have been identified as mobile signals, which are transported through plant vascular tissues to co-ordinate their physiology and development. In this review, we introduce the various grafting techniques that have been developed, we report on the recent advances in the field of plant systemic signaling where grafting techniques have been applied and provide insights for the future.

Identification of phloem-mobile mRNA.

Signaling between cells, tissues and organs is essential for multicellular organisms to coordinate and adapt their development and growth to internal and environmental changes. Plants have evolved a plant-specific symplasmic pathway, called plasmodesmata, for efficient intercellular communication, in addition to the receptor-ligand-based apoplasmic pathway. Long-distance signaling between distant organs is enabled via the phloem tube system, where plasmodesmata contribute to phloem loading and unloading for photosynthate allocation. In addition to signaling by small molecules such as metabolites and phytohormones, the transport of proteins, small RNAs and mRNAs is also considered an important mechanism to achieve long-distance signaling in plants. Recent studies on phloem-mobile proteins and small RNAs have revealed their role in crucial physiological processes including flowering, systemic silencing and nutrient allocation. However, the biological role of mRNAs found in the phloem tube is not yet clear, though their mobility over long-distances has been well evidenced. To gain this knowledge, it is important to collect further information on mRNA profiles in the phloem translocation stream. In this review, I summarize the current approaches to identifying the mRNA population in the phloem translocation system, and discuss the possible role of short- and long-distance mRNA transport.

Plant salt stress status is transmitted systemically via propagating calcium waves

The existence and relevance of rapid long-distance signaling in plants is evident to any observer of the nastic movements of the Venus flytrap (Dionaea muscipula) or the sensitive plant (Mimosa pudica). However, all plants require the transmission of sensory information from the site of perception to other tissues to adjust their physiological states according to their environment. It is becoming increasingly apparent that rapid long-distance signals exist throughout the plant kingdom and may be responsible for initiating a multitude of physiological responses: electrical “action potentials” have been shown to convey wounding and salt-stress information from leaf-to-leaf (1, 2); a “hydraulic signal” transmitted by the direction of water movement within the xylem can mediate long-distance signaling of water stress experienced by the roots to the leaves in Arabidopsis (3); and reactive oxygen species (ROS) have been shown to propagate across a plant and carry stimulus-specific information to a variety of stresses (4). In PNAS, Choi et al. (5) use elegant approaches and present advances demonstrating that calcium can function as a long-distance signaling messenger, propagating in waves from roots and carrying salt-stress signals to induce expression of salt tolerance genes in leaves.

Plant grafting and its application in biological research

Grafting is widely used for various aspects of plant biological research. We reviewed grafting methods and graft development processes for herbaceous plants. We introduced also methods of investigating the development of graft union and compatibility, and the application of grafting in understanding translocation of substances and long-distance signaling in plants.

Alamethicin-induced electrical long distance signaling in plants

Systemic signals induced by wounding and/or pathogen or herbivore attack may be realized by either chemical or mechanical signals. In plants a variety of electrical phenomena have been described and may be considered as signal-transducing events; such as variation potentials (VPs) and action potentials (APs) which propagate over long distances and hence are able to carry information from organ to organ. In addition, we recently described a new type of electrical long-distance signal that propagates systemically, i.e. from leaf to leaf, the ‘system potential’ (SP). This was possible only by establishing a non-invasive method with micro-electrodes positioned in sub-stomatal cavities of open stomata and recording apoplastic responses. Using this technical approach, we investigated the function of the peptaibole alamethicin (ALA), a channel-forming peptide from Trichoderma viride, which is widely used as agent to induce various physiological and defence responses in eukaryotic cells including plants. Although the ability of ALA to initiate changes in membrane potentials in plants has always been postulated it has never been demonstrated. Here we show that both local and long-distance electrical signals, namely depolarization, can be induced by ALA treatment.

Long-distance signaling within Coleus x hybridus leaves; mediated by changes in intra-leaf CO2?

Rapid long-distance signaling in plants can occur via several mechanisms, including symplastic electric coupling and pressure waves. We show here in variegated Coleus leaves a rapid propagation of electrical signals that appears to be caused by changes in intra-leaf CO2 concentrations. Green leaf cells, when illuminated, undergo a rapid depolarization of their membrane potential (Vm) and an increase in their apoplastic pH (pHa) by a process that requires photosynthesis. This is followed by a slower hyperpolarization of Vm and apoplastic acidification, which do not require photosynthesis. White (chlorophyll-lacking) leaf cells, when in isolated white leaf segments, show only the slow response, but when in mixed (i.e. green and white) segments, the rapid Vm depolarization and increase in pHa propagate over more than 10 mm from the green to the white cells. Similarly, these responses propagate 12-20 mm from illuminated to unilluminated green cells. The fact that the propagation of these responses is eliminated when the leaf air spaces are infiltrated with solution indicates that the signal moves in the apoplast rather than the symplast. A depolarization of the mesophyll cells is induced in the dark by a decrease in apoplastic CO2 but not by an increase in pHa. These results support the hypothesis that the propagating signal for the depolarization of the white mesophyll cells is a photosynthetically induced decrease in the CO2 level of the air spaces throughout the leaf.

THE GREAT ESCAPE: Phloem Transport and Unloading of Macromolecules1.

The phloem of higher plants translocates a diverse range of macromolecules including proteins, RNAs, and pathogens. This review considers the origin and destination of such macromolecules. A survey of the literature reveals that the majority of phloem-mobile macromolecules are synthesized within companion cells and enter the sieve elements through the branched plasmodesmata that connect these cells. Examples of systemic macromolecules that originate outside the companion cell are rare and are restricted to viral and subviral pathogens and putative RNA gene-silencing signals, all of which involve a relay system in which the macromolecule is amplified in each successive cell along the pathway to companion cells. Evidence is presented that xenobiotic macromolecules may enter the sieve element by a default pathway as they do not possess the necessary signals for retention in the sieve element-companion cell complex. Several sink tissues possess plasmodesmata with a high-molecular-size exclusion limit, potentially allowing the nonspecific escape of a wide range of small (<50-kDa) macromolecules from the phloem. Larger macromolecules and systemic mRNAs appear to require facilitated transport through sink plasmodesmata. The fate of phloem-mobile macromolecules is considered in relation to current models of long-distance signaling in plants.