Elsevier

Plant Science

Volume 303, February 2021, 110770
Plant Science

Efficient carbon recycling and modulation of antioxidants involved in elongation of the parasitic plant dodder (Cuscuta spp.) in vitro

https://doi.org/10.1016/j.plantsci.2020.110770Get rights and content

Highlights

  • Carbohydrates in the basal stems were continuously degraded as the major storage energy for shoot elongation in vitro.

  • The shoot tips exhibited greater capacity for ROS scavenging compared with the basal stems.

  • Comparative proteomics revealed the different metabolism patterns in the basal stems and the shoot tips.

Abstract

Dodder is a holoparasitic flowering plant that re-establishes parasitism with the host when broken off from the host. However, how in vitro dodder shoots recycle stored nutrients to maintain growth for reparasitizing hosts is not well characterized. Here, the spatial and temporal distribution characteristics of carbohydrates and reactive oxygen species (ROS) were analysed to explore the mechanism of recycling stored nutrients in dodder shoots in vitro. Our results showed that in vitro dodder shoots grew actively for more than 10 d, while dry mass decreased continuously. During this process, the transcript levels and activities of amylases gradually increased until 2 d and then declined in basal stems, which induced starch degradation at the tissue, cellular and subcellular levels. Additionally, the distribution characteristics of H2O2 and the activities and transcript levels of antioxidant enzymes indicated that shoot tips exhibited more robust ROS-scavenging capacity, and basal stems maintained higher ROS accumulation. Comparative proteomics analysis revealed that starch in basal stems acted as an energy source, and the glycolysis, TCA cycle and pentose phosphate pathway represented the energy supply for shoot tip elongation with time. These results indicated that efficient nutrient recycling and ROS modulation facilitated the parasitism of dodder grown in vitro by promoting shoot elongation growth to reach the host.

Introduction

Dodders (Cuscuta spp.) are holoparasitic plants that possess parasitism-specific organ haustoria, which connect to host vascular bundles for extracting water, nutrients and metabolites [1,2]. Dodder appears to lack leaves, and some species have lost extensive portions of their plastid genomes to be functionally non-photosynthetic, although others synthesize chlorophyll [3]. Modelling the flow and partitioning of carbon between dodder and the host Lupinus albus revealed that dodder derived 81 % of photosynthate from Lupinus albus [4]. Dodder, as an obligate stem parasite, possesses a different strategy. The dormancy of dodder seeds, which may last for decades, is independent of the presence of the host [1]. After germination, dodder has to find a host within several days; otherwise, it dies [5]. Rather than randomly searching for a host plant, in vitro dodder seedlings are highly attuned to sensing the location of the host guided by volatile chemicals emitted by the host [6]. However, host plants are not always located in close proximity from germinating in vitro dodder seedlings; thus, dodder seedlings must quickly extend the length of the thread-like stem to reach the host. Therefore, finding a host is an essential issue for continuing the lifecycle at the in vitro stage of dodder. In general, up to 66 % of dodder shoots naturally break off from the host due to physical forces such as wind and animals [7,8]. These fragmentations do not wither away quickly and try to reparasitize a host [8,9]. Nevertheless, the biological basis of in vitro dodder shoot reparasitism is still not well characterized.

It is well known that the ability to continuously sense and control energy status is vital for surviving under stress [10]. The effective distribution and allocation of carbohydrates allow plants to adapt to various stresses, such as drought, chilling, nutrition starvation and attacks by pathogens [11]. This adaptation is often accompanied by changing large numbers of stress-responsive genes by sugar signalling, which indicates the role of sugars in plant responses to various environmental conditions [[12], [13], [14]]. In plants, the availability of sugars is crucial for development since sugars act as both immediate substrates for metabolism and signalling molecules [15]. Meanwhile, starch, as the most abundant carbohydrate reserve in plants, can instead represent an adequate carbon supply for several days and can play pivotal roles in ensuring the rapid induction of genes encoding enzymes in response to sugar starvation [16]. In addition, soluble sugars act as nutrient and metabolite signalling molecules, activating specific pathways to regulate plant sugar status [17]. As dodder does not employ photosynthesis and in the absence of an energy source from host plants, it is very important for dodder to reuse stored carbohydrate assimilates serving as an energy source for in vitro shoot growth. However, little information is available regarding the involvement of sugar nutrient and metabolite signalling molecules in modulating the growth of in vitro dodder shoots.

Sugar metabolism and signalling function in an intricate network with reactive oxygen species (ROS) production, signalling and scavenging systems [18]. ROS are collections of several types of active molecules, including O2radical dot, H2O2, OH radical dotand 1O2, which are inevitable products of normal plant growth and metabolism [19]. The lifetime of ROS is operated largely by a complex antioxidant network that consists of antioxidant enzymes such as catalase (CAT), ascorbate peroxidase (APX), and the other enzymes of the ascorbate-glutathione cycle and low molecular antioxidants such as ascorbate, glutathione and flavonoids [20]. Sugar availability can feed the oxidative pentose phosphate pathway by increasing the reducing power for glutathione production and contributing to H2O2 scavenging [17]. By generating glucose-6-phosphate, hexose kinases (HXKs) can stimulate the biosynthesis of ascorbic acid, which has major roles in the ROS scavenging process [21]. ROS are common molecules in various plant metabolic processes and were once thought to be harmful to plants because excessive accumulation might lead to oxidative stress. However, a timely and appropriate burst of ROS can act as an important signal for plant growth, development and defence against external stresses [22]; for example, ROS play a crucial role in the transition from proliferation to differentiation in root growth [23]. In addition, ROS function in cells as signalling molecules to monitor different metabolic reactions in plants, animals, and eukaryotic organisms [24].

Dodder as a dominant sink, acquires most of its carbohydrates from its host plants [25]. Although there is a lack of carbon supply from host plants, excised dodder shoots can grow for several weeks and reparasitize new hosts if available (Fig. S1). In general, nutritional deficiency may accelerate senescence and assimilate the transportation of plants, and metabolic changes during senescence are essential to support active growth in younger tissues [26,27]. Meanwhile, senescence can alter plant source-sink relationships, e.g., the nutrient content of sink organs in crop plants, such as seeds, comes from the assimilation of the photosynthetic apparatus and transport from the senescence of vegetative tissues [28]. For in vitro growth of dodder shoots, in low energy status, the morphologically basal stems changed into source organs that exported the reserved energy to sustain shoot tip growth, which acted as sink tissue in the source-sink relationship of dodder shoots. However, the physiological and molecular characteristics of carbohydrate nutrient recycling in in vitro dodder shoots are largely unknown.

In this study, the temporal and spatial distribution characteristics of starch and soluble sugars were analysed in the different segments of in vitro dodder shoots, and carbohydrate metabolism-related genes were detected. Meanwhile, the timing of ROS occurrence was determined, accompanied by the measurement of ROS scavengers in in vitro dodder shoots. Moreover, comparative proteomics analysis was used to evaluate the differentially expressed proteins involved in in vitro dodder shoot development, which would be beneficial for understanding the possible signalling pathways involved in this biological process. These results provide fundamental knowledge for resisting in vitro dodder attacks and the regulation of sources and sinks in plants.

Section snippets

Plant material and growth conditions

Cuscuta suaveolens seed was a gift from Dr P. Reisen at Forage Genetics International, Indiana. The C. suaveolens seeds were treated with concentrated H2SO4 for 30 min to break seed dormancy, germinated in petri dishes containing two layers of wet filter paper with deionized water and then incubated in a growth chamber at 25 °C under 16 h light/8 h dark. Alfalfa (Medicago sativa) seeds were grown in plastic pots containing a 1:4 mixture of vermiculite and nutrient soil moistened with water.

Morphological and cytological characteristics in the growth of dodder shoots in vitro

After isolation from the host, in vitro dodder shoots continued growing and extended from 7 cm at 0 d to 16 cm at 10 d (Fig. 1A, Fig. S2). The growth rate, indicated by the slope, was highest from 0 to 2 d and then gradually declined from 2 to 10 d. Conversely, with the increase in length, the total dry mass of dodder shoots decreased continuously, and the net dry mass decreased by 34.4 % at 10 d in vitro (Fig. 1B). To understand the tissue-specific physiology changes in this process, the

Discussion

Dodder is a holoparasitic plant because its photosynthetic ability has been greatly degenerated, so in vitro dodder shoots have to rely primarily on their own carbon reserves for elongation. Excised dodder shoots, unlike other plants, exhibited rapid growth and elongation for several days (Fig. 1A). This process was mediated by source-sink communication, in which the basal stems acted as carbon supply and the shoot tips acted as carbon demand. During growth in vitro, dodder shoots experienced

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by the National NaturalScience Foundation of China (31272055). The authors also thank Dr. Calvin G. Messersmith (North Dakota State University, USA) and Dr. Douglas James Doohan (Ohio State University, USA) for technical and linguistical improvement of the manuscript.

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