Keywords

10.1 Introduction

In the natural environment, plants are often bombarded by abiotic (such as drought, salt, heat or cold) and biotic (necrotrophic and biotrophic pathogens) stresses (Ku et al. 2018; Kollist et al. 2019). The negative effects of environmental stresses, including abiotic and biotic ones, significantly decrease crop productivity (Anwar et al. 2018). Biotic stress is a significant reason for crop loss and causes yield losses in the range of 31–42%, post-harvest loss due to biotic stress is in the range of 6–20%, and abiotic stress causes 6–20% of the crop damage (Shameer et al. 2018). During the evolution, plants have developed complex strategies that regulate plant developmental, morphological, biochemical and physiological acclimation in order to respond to biotic and abiotic stress (Yongxue et al. 2016; Jian Kang 2016). With the rise of molecular technologies, the mechanisms of plants response to biotic and abiotic stresses including the gene expression patterns, protein patterns and metabolite patterns can be efficiently analyzed by a combination of molecular techniques including genomics, transcriptomics, transgenic technologies, proteomics and metabolomics (Yongxue et al. 2016; Voelckel et al. 2017; Silveira and Carvalho 2016). Recognizing the molecular mechanisms driving plant stress related events and developing molecular strategies to aid plants to tolerate, resist or adapt to biotic and abiotic stress are critical for improving stress tolerance in plants.

Sugar beet (Beta vulgaris L.) is a typical vegetative crop with a biennial life cycle, which has become an important source for sugar production in temperate areas of the world (Hossain et al. 2017). Sugar beet is cultivated in different climates in Europe, North America, and increasingly in Asia, South America and recently North Africa, which suggests that sugar beet cultivars have certain adaptability to different environments and growth conditions (Hossain et al. 2017; Vastarelli et al. 2013). Sugar beet is moderately salt-tolerant and has certain resistance to cold and drought stress. It is not only used in sugar production, but also used in a wide range of industries including the production of bioethanol as a source of renewable energy (Valli et al. 2012), extraction of pectin from sugar beet pulp (Lv et al. 2013) and production of molasses used as antioxidants, sweetener and colorant (Valli et al. 2012). The root vegetable Beta vulgaris rubra, otherwise known as red beetroot (referred to as beetroot) has attracted much attention as a health-promoting and disease-preventing functional food (Clifford et al. 2015). In Central and Eastern Europe, red beetroot is a popular vegetable, and commonly used in food industry as a coloring agent (Georgiev et al. 2010). Known for a long time for its beneficial health effects, beetroot and red beetroot extracts possess antihypertensive, hypoglycemic, antioxidant, anti-inflammatory and hepatoprotective activities and is an effective multiorgan tumor suppressing agent in laboratory animals (Sulakhiya et al. 2016; Krajka-Kuźniak et al. 2012; Gezginci-Oktayoglu et al. 2014).

Sugar beet as a cash crop requires careful agronomical practices and breeding for adaptation to biotic and abiotic stresses to maintain its profitability and ability to produce high-quality taproots. The genome sequence of sugar beet was reported by Dohm et al (2014). In this paper, we review the mechanisms of sugar beet response to biotic and abiotic stresses at the levels of physiological change, the genes’ functions, transcription and translation. Understanding how sugar beet responds and tolerates biotic and abiotic stresses is important for boosting sugar beet productivity under these challenging conditions. In order to minimize the negative impact of these stresses, studying how sugar beet has evolved stress coping mechanisms will provide new insights and lead to novel strategies for improving the breeding of stress-resistant sugar beet and other crops.

10.2 The Morphological and Physiological Changes of Sugar Beet in Response to Biotic and Abiotic Stresses

Biotic and abiotic stresses affect plant phenotype and physiology. Salt and drought are the main abiotic stresses in sugar beet that have been extensively studied. The phenotypic and physiological changes of sugar beet under salt, drought and various stress conditions in recent years have been summarized (Table 10.S1). The stress tolerance of sugar beet is a complex trait determined by many physiological and metabolic pathways. These results have laid a foundation for understanding the physiological regulatory mechanisms of stress tolerance in Beta vulgaris.

Table 10.S1 The physiological changes of sugar beet under stress conditions
Full size table

10.2.1 The Morphological and Physiological Changes Under Salt Conditions

Fine-tuned and coordinated regulation of transport, metabolism and redox homeostasis allows plants to acclimate to osmotic and ionic stress caused by high salinity. Sugar beet (Beta vulgaris ssp. vulgaris), cultivar KWS2320 plants were subjected to a final level of 300 mM NaCl for up to 14 days in hydroponics (Hossain et al. 2017). Sugar beet monosomic addition line M14 tolerates salinity of up to 500 mM sodium chloride (NaCl) for 7 days without losing viability (Yang et al. 2012). Sugar beet is a highly salt tolerant crop plant and is therefore an interesting model to study sodium chloride (NaCl) acclimation in crops.

Some of the central components of the cellular antioxidant defense system in sugar beet were identified, and followed their transcriptional response during acclimation to 300 mM NaCl (Hossain et al. 2017).

The salt tolerance of sugar beet was integrated into many aspects, including storing salt ions in old leaves and petioles, increasing the levels of compatible solutes, enhancing the activity of antioxidants and increasing NADP-ME, NADP-MDHN and PEPC activities under moderate salt stress (Wang et al. 2017).

The effect of saline-alkali stress on dry mass, rhizosphere soil enzyme activities and soil microbial quantities were recorded (Guo et al. 2016). The results showed that the numbers of soil microbial communities and the activities of soil enzymes directly affected the stress resistance of sugar beet.

The causal link between mesophyll ion transport activity and plant salt tolerance in a range of evolutionary contrasting halophyte (Vicia faba L., Broad beans; Beta vulgaris L., Sugar beet; Pisum sativum L., Pea) and glycophyte species (Chenopodium quinoa, Quinoa; Mesembryanthemum purpureus, Pigface; Disphyma crassifolium, Noon-flower) were established (Percey et al. 2016). Accordingly, whole-plant physiological and agronomical performance also clearly followed quinoa > sugar beet > bean pattern. In sugar beet, photosynthetic parameters under salinity stress showed no decline. Moreover, in sugar beet the chlorophyll content even increased significantly in plants grown at high NaCl concentrations which was attributed to the ability of mesophyll cells to control cytosolic K+ homeostasis under the salinity stress conditions.

The effect of different Na+/K+ ratios on plasma membrane (PM) H+-ATPase activity in salt sensitive maize (Zea mays L.) and salt-resistant sugar beet shoot were researched in vitro (Wakeel et al. 2011a). An increased Na+/K+ ratio decreased the PM H+-ATPase activity in vesicles from maize as well as from sugar beet shoots. Nevertheless, the detrimental effect of an increased Na+/K+ ratio was more severe in salt-sensitive maize compared to salt-resistant sugar beet. Sugar beet expresses specific H+-ATPases, which were more tolerant of elevated Na+ concentration as compared to those of salt-sensitive maize. Plant growth is impaired primarily by osmotic stress in the first phase of salt stress (Munns 1993), whereas Na+ toxicity affects the plant growth mainly in the second phase (Fortmeier and Schubert 1995; Sümer et al. 2004). Salinity leads to an increased Na+/K+ ratio and thus displacement of K+ by Na+ in the plant cell. Relatively higher cytosolic Na+ concentrations may have an effect on the activity of plasma membrane (PM) H+-ATPase. A decreased PM-H+-ATPase activity could increase the apoplastic pH (Bibikova et al. 1998). This process could limit the cell-wall extensibility and thus reduce growth according to the acid growth theory (Rayle and Cleland 1992; Cosgrove 1997). So, when cytosolic Na+ accumulation occurs, specific Na+-tolerant ATPase isoforms may contribute to salt resistance of sugar beet.

Utilization of rhizobacteria associated with plant roots in harsh environments could be a feasible strategy to deal with limits to agricultural production caused by soil salinity (Timmusk et al. 2014). Halophytes occur naturally in high-salt environments, and their roots may be associated with promising microbial candidates for promoting growth and salt tolerance in crops (Jha et al. 2012; Zhao et al. 2016). The efficient halotolerant plant growth-promoting rhizobacterial strains from halophytes were isolated and their activity and effects on sugar beet growth were evaluated under salinity stress (Zhou et al. 2017). Three isolates, identified as Micrococcus yunnanensis, Planococcus rifietoensis and Variovorax paradoxus enhanced salt stress tolerance remarkably in sugar beet, resulting in greater seed germination and plant biomass, higher photosynthetic capacity and lower stress-induced ethylene production at different NaCl concentrations (50–125 mM). These results demonstrated that salinity adapted 1-aminocyclopropane-1-carboxylate deaminase (ACD)-producing bacteria isolated from halophytes could promote sugar beet growth under saline stress conditions.

10.2.2 The Morphological and Physiological Changes Under Drought Conditions

Leaf vascular bundles and palisade tissues of sugar beet are well developed, and the species is a direct root crop with strong drought tolerance (Li et al. 2016). Root yield, sucrose content and juice purity are essential for the efficiency of sugar manufacturing and have been continuously improved by breeding. To ensure further progress, however, tolerance against biotic and abiotic stress factors such as drought is gaining increasing importance (Jansen and Stibbe 2007). In recent years, drought has been found to be one of the major factors limiting sugar beet yield (Pidgeon et al. 2005).

Sugar beet (Beta vulgaris cv. Beta 356) was subjected to drought stress during the vegetative development by maintaining the soil water content in the 0–40 cm soil depth at 70%, 50% or 30% of field capacity to study the physiological traits of the leaves (Li et al. 2016). The results showed that the compensation index was at the highest in the 50% field capacity treatment. Supplemental irrigation should be carried out promptly when the soil water content drops to 50% of field capacity during vegetative development. Rehydration could promote self-repair functions in leaves, thus reducing the effects of drought on sugar beet yield and sugar content.

Mutation induction with gamma radiation via in vitro tissue culture is a useful technique for improving plant tolerance against environmental stresses. Putative drought-tolerant sugar beet plants were obtained in in vitro tissue cultures via mutation induction using 20 Gy gamma irradiation. The changes in the antioxidant enzyme systems that were detected both spectrophotometrically and on the level of isoenzyme variations in the earlier stages of development have the ability to determine the differences between the mutant plants and the control. It is hoped that the data presented here will be valuable for future studies on genotype selection, plant development and characterization of gene sources in sugar beet (Sen and Alikamanoglu 2014).

10.2.3 The Morphological and Physiological Changes Under Other Stress Conditions

The oxidative damage caused by light stress in plants and the consequent involvement of pigments are widely studied. The nuclear magnetic resonance (NMR) spectroscopy can be utilized to compare crude leaf extract at different levels of light stress, allowing the analysis of these compounds (Tiziani et al. 2006). The analysis of the 1H NMR (1D) spectra of two agronomic species (Spinacia oleracea and Beta vulgaris) exposed to different light intensities was presented (Guadagno et al. 2013). In particular, changes in carotenoids and xanthophylls signals were analyzed. NMR spectroscopy provides a powerful complementary way for the identification and quantitative analysis of sugar beet and other plant metabolites either in vivo or in tissue extracts induced by light stress.

Phenol-dependent (guaiacol) peroxidase (POD) is a well-known antioxidant enzyme that protects plant cells from oxidative damage. The activity of POD and its isozyme composition at different stages of the red beet root development were investigated and simultaneously the activity and isozyme composition of POD under hyperosmotic stress and infection of roots by microorganisms were analyzed (Pradedova et al. 2014). The data showed that the activity and isozyme composition of the vacuolar POD, similarly to PODs of other cell compartments changed at different stages of sugar beet development and under stress conditions. So, the changes in the activity and isozyme composition of the vacuolar POD suggest that the central vacuole is not only actively involved in the metabolic processes associated with plant growth and development but is also an important component of cell defense in different types of stress.

The establishment of stress resilient sugar beet is an important breeding goal since this cash crop is susceptible to drought and salinity. The genetic diversity in cultivated sugar beets is low and the beet wild relatives are useful genetic resources for tolerance traits (Fénart et al. 2008; Panella and Lewellen 2007). Three wild beet populations (Beta vulgaris ssp. maritima) from contrasting environments, Vaiamonte (VMT, dry inland hill), Comporta (CMP, marsh) and Oeiras (OEI, coastland), and one commercial sugar beet (Isella variety, SB), were compared. At the genetic level, the use of six microsatellite allowed to detect a total of 76 alleles. The physiological responses of the populations under drought and salt stress, namely at initial stress stages, late stress stages, and early stress recovery were evaluated. Two striking ecotypes were VMT, which was the best to cope with drought and salinity, and CMP which had the highest root to shoot ratio (Ribeiro et al. 2016). These genotypes can supply breeding programs with distinct goals.

10.2.4 The Tolerance and Adaptation Mechanisms in Physiological Changes of Sugar Beet in Response to Biotic and Abiotic Stresses

Reactive oxygen species (ROS) accumulation is a common denominator under conditions of stress (Foyer et al. 1994). Complementary to the generator systems are enzymatic and non-enzymatic antioxidants that regulate ROS and redox homeostasis and counteract metabolic imbalances, damage to cell structures, cell death and stress adaptation (Foyer et al. 2009). It was concluded that the increase in the content of enzymes and non-enzymatic antioxidants were also the main strategies for sugar beet to regulate ROS and redox homeostasis under salt, drought and various stresses.

The ability of maintaining a stable Na+/K+ ratio and cytosolic K+ homeostasis were the main factors in the sugar beet response to salt stress. The increase in proline is one of the main physiological indicators of sugar beet response to drought stress. The stress tolerance potential of sugar beet was improved by crossbreeding with wild type sugar beet, mutation breeding and inoculating rhizobacteria from the rhizosphere of halophytes to the root of sugar beet.

10.3 The Functions of Genes from Sugar Beet in Response to Stresses

Transgenic technology is an important method to study gene functions. Recently, studies on the function of stress-responsive genes in sugar beet were mainly focused on salt stress. The functions of genes responding to cold and heavy metal stress have also investigated. These genes in sugar beet will provide potential candidate genes for improving stress resistance in sugar beet and other crops.

10.3.1 The Functions of Genes from Sugar Beet in Response to Salt Stress

Sugar beet is an economically important, salt-tolerant, and widely distributed plant, which can grow under 300 mM NaCl and even 500 mM treatment (Hossain et al. 2017; Yang et al. 2012). It accumulates glycine betaine (GB) as an osmoprotectant under NaCl stress and is an excellent model plant to study the mechanisms of salt stress. Several salt-stress-related genes have been isolated from sugar beet (Russell et al. 1998; Tabuchi et al. 2006).

Serine biosynthesis in plants proceeds through two pathways: the phosphorylated pathway and the photorespiratory pathway (Ros et al. 2014). In the photorespiratory pathway, serine is synthesized from glycine in the presence of serine hydroxymethyl transferase (SHMT) (Ros et al. 2014). In the phosphorylated pathway, serine is derived from 3-phosphoglycerate (3-PGA). D-3-phosphoglycerate dehydrogenase (PGDH) catalyzes the oxidation of 3-PGA to form phosphohydroxypyruvate (PHP) by utilizing NAD as a cofactor (Ros et al. 2014). One study investigated the significance of serine biosynthetic genes for salt stress in sugar beet (Kito et al. 2017). mRNA transcriptional expression for BvPGDHa was significantly enhanced under salt stress conditions in both leaves and roots of sugar beet. On the other hand, BvSHMTa was expressed transiently in leaves and roots under salt stress. PGDH activity was high in storage root. After salt stress, PGDH activity was increased in leaf, petiole, and root. Recombinant proteins were expressed in Escherichia coli. The results suggest that BvPGDHa and BvSHMTa play an important role in salt tolerance.

Sugar beet accumulates large amount of GB in the leaves as well as in the storage roots (Hanson and Wise 1982). GB is an important osmoprotectant and is synthesized by two-step oxidation of choline (Takabe et al. 2006). Choline monooxygenase (CMO) catalyzes the first step of the pathway and is believed to be a rate-limiting step for GB synthesis (Rathinasabapathi et al. 1997). Since many crop plants do not have the GB biosynthetic pathway, genetic engineering of this pathway could be useful to improve stress tolerance (Rontein et al. 2002). In order to investigate the role of CMO for GB accumulation in sugar beet, transgenic plants carrying the antisense BvCMO gene were developed (Yamada et al. 2015). The antisense BvCMO plants showed the decreased activity of GB synthesis from choline compared to wild-type (WT) plants, which was well related to the suppressed level of BvCMO protein. However, GB contents were similar between transgenic and WT plants with the exception of young leaves and storage roots. Transgenic plants showed enhanced susceptibility to salt stress compared with WT plants. These results suggest the importance of choline precursor supply for GB accumulation, and young leaves and storage root as sensitive sites for GB accumulation.

Na+/H+ exchange (NHX) activity was correlated with the salinity tolerance of B. vulgaris cell cultures (Blumwald and Poole 1987). A cDNA encoding BvNHX1 cation exchanger protein was isolated (Xia et al. 2002). The BvNHX1 transcript levels in both the suspension cell culture and whole plants increased following salt treatment and this increase was concomitant with the elevated BvNHX1 protein and vacuolar Na+/H+ antiporter activity (Xia et al. 2002). The promoter of BvNHX1 was cloned and its activity was studied in transgenic Arabidopsis expressing the BvNHX1::GUS construct (Adler et al. 2010). The minimal 337 bp promoter sequence that was upregulated by salt treatment and osmotic stress was identified by constructs containing serial deletions of the promoter. Mutating four putative cis-acting elements within the 337 bp promoter fragment revealed that MYB transcription factor(s) is involved in the activation of the expression of BvNHX1 upon exposure to salt and water stresses.

Sugar beet M14 line is an interspecific hybrid crossed between cultivated sugar beet B. vulgaris L. and wild species B. corolliflora Zoss., which contains the 9th chromosome of B. corolliflora and the complete B. vulgaris genome (Guo et al. 1994). It has several interesting characteristics including apomixis and tolerance to drought, cold and salt stress (Yang et al. 2012). Sugar beet M14 therefore can function as a unique germplasm for isolating genes from the wild species for improvement in cultivated beet. The functions of three genes (BvM14-glyoxalase I, BvM14-cystatin and BvM14-SAMS2) in response to salt stress were studied in sugar beet M14.

Methylglyoxal (MG) is a by-product of several metabolic pathways that are involved in the degradation of carbohydrates, proteins and lipids (Hoque et al. 2016). However, under environmental stresses, both biotic and abiotic, MG production is abruptly increased making its levels highly toxic to cellular biomolecules, including proteins, DNA, RNA and lipids (Bilova et al. 2017). To combat MG toxicity, plants contain a powerful glyoxalase system comprising glyoxalase I and II, the enzymes that detoxify excessive MG using glutathione (GSH) as co-factor (Kaur et al. 2017; Ghosh et al. 2014). The BvM14-glyoxalase I gene from sugar beet M14 is ubiquitously expressed in different tissues of sugar beet M14 line and upregulated in response to salt, mannitol and oxidative stresses. Heterologous expression of BvM14-glyoxalase I increases E. coli tolerance to methylglyoxal. Transgenic tobacco plants constitutively expressing BvM14-glyoxalase I were generated. Both leaf discs and seedlings showed significant tolerance to methylglyoxal, salt, mannitol and H2O2. These results suggest an important role of BvM14-glyoxalase I in cellular detoxification and tolerance to abiotic stresses.

Constitutive expression of Arabidopsis cystatins in yeast and Arabidopsis seedlings increased their tolerance to high salt, drought, oxidative, and cold stresses (Zhang 2008). Overall, cystatins may play a crucial role in plant stress tolerance and defense (Van der et al. 2003). BvM14-cystatin from sugar beet M14 was expressed ubiquitously in roots, stems, leaves and flower tissues with relatively high abundance in developing stems and roots (Wang et al. 2012). It was found to be localized in the nucleus, cytoplasm and plasma membrane. Recombinant BvM14-cystatin expressed in E. coli was purified and exhibited cysteine protease inhibitor activity. Salt-stress treatment induced BvM14-cystatin transcript levels in the M14 seedlings. Homozygous Arabidopsis plants over-expressing BvM14-cystatin showed enhanced salt tolerance. Taken together, these data improved understanding of the functions of BvM14-cystatin and highlighted the possibility of employing the cystatin in engineering plants for enhanced salt tolerance.

S-adenosyl-L-methionine synthetase (SAMS), one of the salt-responsive genes, is an important enzyme in the synthesis of SAM. Usually, SAM synthesized by SAMS from methionine and ATP acts as a universal methyl group donor involved in numerous transmethylation reactions (Roje 2006). It plays a vital role in metabolism and development regulation, as well as in abiotic and biotic stress responses (Nagel et al. 2008; Köllner et al. 2010). Additionally, it functions as a precursor for the synthesis of polyamines (PAs), which are involved in regulating plant responses to abiotic or biotic stresses (Jang et al. 2012). To determine the potential role of SAMS from sugar beet in salt tolerance, BvM14-SAMS2 gene from the salt-tolerant sugar beet M14 was isolated (Ma et al. 2017). The expression of BvM14-SAMS2 in leaves and roots was greatly induced by salt stress. Overexpression of BvM14-SAMS2 in Arabidopsis resulted in enhanced salt and H2O2 tolerance. Furthermore, a knock-down T-DNA insertion mutant of AtSAMS3, which shared the highest homology with BvM14-SAMS2, was obtained Interestingly, the mutant atsam3 showed sensitivity to salt and H2O2 stress. The result showed that the antioxidant system and polyamine metabolism played an important role in salt and H2O2 tolerance in the BvM14-SAMS2 overexpressed plants. The results had provided new insights into SAMS functions in sugar beet.

Plants acclimate to NaCl stress by maintaining its growth rate and adjusting its cellular redox and ROS network. In order to understand the mechanisms of ROS accumulation and scavenging in sugar beet under severe salinity, the regulation of key enzymatic antioxidants involved in the redox and ROS network was investigated at the transcript level. Sugar beet plants were subjected to a final level of 300 mM NaCl for up to 14 days in hydroponics (Hossain et al. 2017). First, the gene families of superoxide dismutase (SOD), peroxiredoxins (Prx), alternative oxidase (AOX), plastid terminal oxidase (PTOX) and NADPH oxidase (RBOH) were identified in the sugar beet genome (Hossain et al. 2017). Salinity induced the accumulation of Cu-Zn-SOD, Mn-SOD, Fe-SOD3, all AOX isoforms, 2-Cys-PrxB, PrxQ, and PrxIIF at the transcriptional level (Hossain et al. 2017). In contrast, Fe-SOD1, 1-Cys-Prx, PrxIIB and PrxIIE levels decreased in response to salinity at the transcriptional level. Most importantly, RBOH transcripts of all isoforms decreased (Hossain et al. 2017). The effect of salt stress on BvpAPX expression in leaves of the cultivated beet varieties, Huzar and Janosik, and their wild salt-tolerant relative B. vulgaris ssp. maritima were determined (Dunajska et al. 2014). Plants were subjected to salt stress during a 32-day culture period (long-term salt treatment). An alternative salinization protocol consisted of an 18-h incubation of detached beet leaves in media supplemented with toxic salt concentrations (short-term salt treatment). qRT-PCR analysis revealed that BvpAPX expression markedly increased in leaves of plants subjected to conditions of long-term treatment with salinity, whereas BvpAPX transcript levels remained unaffected in detached leaves during short-term salt treatment. It is possible that assessment of the timing of transcriptional activation of the BvpAPX gene in plants challenged with long-term treatments with salinity may provide cues for screening beet genotypes for salt tolerance.

10.3.2 The Functions of Genes from Sugar Beet in Response to Cold Stress and Heavy Metal Stress

An early response to dehydration like 6 protein (ERDL6) in Arabidopsis was demonstrated directly to operate as a proton-driven glucose exporter involved in glucose export from vacuoles into the cytosol (Poschet et al. 2011). Overexpression of BvIMP, the closest sugar beet homolog to AtERDL6, in Arabidopsis affected glucose concentrations under cold conditions and led, surprisingly, to impaired seed germination under both conditions, sugar application and low environmental temperatures (Klemens et al. 2014). Upon cold treatment, BvIMP overexpressing plants accumulated lower quantities of monosaccharides than the wild-type plants, a response in line with the reduced frost tolerance of the transgenic Arabidopsis plants, and the fact that cold temperatures inhibited BvIMP transcription in sugar beet leaves. These findings showed that the tight control of vacuolar sugar import and export was a key requisite for cold tolerance and seed germination of plants.

Sugar beet is an emerging renewable energy crop with high biomass and ethanol conversion rate and a strong ability to adapt to the environment, making it a suitable target species for phytoremediation which is a highly efficient, economical and environmentally friendly way to remove heavy metals from contaminated soil (Reeves and Baker 2000). During the remediation process, these toxic elements are extracted or stabilized by plants and metabolized in their tissues (Reeves and Baker 2000). A novel Streptococcus thermophiles γ-glutamylcysteine synthetase-glutathione synthetase (StGCS-GS) that synthesizes GSH with limited feedback inhibition was overexpressed in sugar beet, yielding three transgenic lines with enhanced tolerance to different concentrations of cadmium, zinc and copper, as indicated by their increased biomass, root length and relative growth compared with wild-type plants (Liu et al. 2015). Transgenic sugar beets accumulated more Cd, Zn and Cu ions in shoots than wild-type, as well as higher GSH and phytochelatin (PC) levels under different heavy metal stresses. This enhanced heavy metal tolerance and increased accumulation were likely due to the increased expression of StGCS-GS and consequent overproduction of both GSH and PC. The results demonstrate the explicit role of StGCS-GS in enhancing Cd, Zn and Cu tolerance and accumulation in transgenic sugar beet, which may represent a highly promising new tool for phytoremediation.

10.3.3 The Molecular Mechanisms of Genes from Sugar Beet in Response to Stresses

The functions of 15 genes related to sugar beet in response to salt, cold and heavy metal stresses were summarized by transgenic technologies or the transcriptional expression level, providing a good basis for further exploitation and utilization of stress-resistant gene resources in sugar beet.

10.4 The Transcriptome Analysis of Sugar Beet in Response to Biotic and Abiotic Stresses

Plant transcriptomics is widely used in studying plant responses to various stresses. Transcriptomic studies have revealed many changes in expression levels of various genes during exposure to environmental extremes (Imadi et al. 2015). Transcriptomics are applied widely in plants to help us understand physiological and molecular responses in terms of genome sequence, gene regulation, gene differentiation, posttranscriptional modifications and gene splicing and are used to quantify the modulations in gene expression levels during different stress conditions and developmental stages (Wang et al. 2009). Sugar beet transcriptome have recently received some impulse, which have focused on exposure to multi-stress conditions including salt, cold and other stresses.

10.4.1 The Transcriptome Analysis of Sugar Beet in Response to Salt Stress

Previous studies reported that sugar beet monosomic addition line M14 obtained from the intercross between Beta vulgaris L. (cultivated species) and B. corolliflora Zoss (wild species) exhibited tolerance to salt (up to 0.5 M NaCl) stress (Yang et al. 2012). Comparative transcriptomics was performed to monitor genes differentially expressed in the leaf and root samples of the sugar beet M14 seedlings treated with 0, 200 and 400 mM NaCl, respectively (Lv et al. 2018). Digital gene expression revealed that 3856 unigenes in leaves and 7157 unigenes in roots were differentially expressed under salt stress. Enrichment analysis of the differentially expressed genes based on GO and KEGG databases showed that in both leaves and roots genes related to regulation of redox balance, signal transduction, and protein phosphorylation were differentially expressed. Comparison of gene expression in the leaf and root samples treated with 200 and 400 mM NaCl revealed different mechanisms for coping with salt stress. In addition, the expression levels of nine unigenes in the ROS scavenging system exhibited significant differences in the leaves and roots. The transcriptomics results have provided new insights into the salt-stress responses in the leaves and roots of sugar beet.

Sea beet (Beta vulgaris ssp. maritima) is a wild relative of cultivated beets (Beta vulgaris) which displays elevated salt stress-resistance, compared to other beet varieties. It is reported that sea beet has higher osmotic adjustment value and higher leaf succulence index compared to the cultivated beets (Bagatta et al. 2008). The transcriptome response to acute salt stress imposed to excised leaves of sea beet was investigated (Skorupa et al. 2016). Sequencing libraries were generated from leaves subjected to either moderate (150 mM NaCl) or strong (300 mM NaCl) salt stress. Control libraries were constructed from untreated leaves. Sequencing was performed using the Illumina MiSeq platform. 32,970 unigenes were obtained by assembling the pooled reads from all the libraries with Trinity software. Screening the nr database returned 18,362 sequences with functional annotation. Using the reference transcriptome, 1246 genes that were differentially expressed after 48 hours of NaCl stress were identified. Genes related to several cellular functions such as membrane transport, osmoprotection, molecular chaperoning, redox metabolism or protein synthesis were differentially expressed in response to salt stress. The response of sea beet leaves to salt treatments was marked out by transcriptomic upregulation of genes related to photosynthetic carbon fixation, ribosome biogenesis, cell wall building and cell wall expansion.

10.4.2 The Transcriptome Analysis of Sugar Beet in Response to Cold Stress

The reference transcriptome assembly has been published for sugar beet adult plants under gibberellin treatment and vernalization (Mutasa-Go¨ttgens et al. 2012) and for elucidating the pathway of accumulation of sucrose in taproots (Turesson et al. 2014).

Low-temperature stress is a significant cause of sugar beet quality and production losses in European and North American agriculture (Kirchhoff et al. 2012). The survival of young sugar beet plantlets and the subsequent sucrose yield of mature plants are often seriously limited by low temperatures, especially when plantlets are exposed to freezing temperatures (below 0 °C) at early developmental stages (Kirchhoff et al. 2012). The wide study of sugar beet transcriptome modulation after a short exposure to a cold stress, mimicking what was experienced in vivo by young plantlets when temperature droped in the early spring nights, was carried out by high-throughput sequencing of leaves and root RNAs (RNA-Seq) (Moliterni et al. 2015). In total, approximately 200 million of Illumina paired-end reads were obtained and 20,927 unique contigs with a read count of at least 100 reads per contig were assembled, with an average length of 2161 bp. When comparing the transcriptome of control and low temperature-treated sugar beet plants, a total of 549 (451 positively and 98 negatively regulated) differentially expressed sequences in leaves and 656 (555 positively and 101 negatively) in roots were retrieved. A significant picture of the earliest events of temperature sensing was achieved for the first time for sugar beet: the retrieval of a great amount of transcription factors and the intensity of modulation of a large number of genes involved in several metabolic pathways suggested a fast and deep rearrangement of sugar beet plantlets metabolism as early response to cold stress, with both similarities and specificities between the leaf and root tissues.

10.4.3 The Transcriptome Analysis of Sugar Beet in Response to Biotic Stress

Suppressive subtractive hybridization (SSH) is a technique used to identify differentially expressed genes in cells important for growth and differentiation at the transcriptome level, which has often been used to study molecular mechanisms of plants in biotic and abiotic stresses (Lukyanov et al. 2007; Sahebi et al. 2015). The response to insect pests in the root of sugar beet is an interesting area of plant defense research. More than 150 sugar beet root ESTs (Expressed sequence tags) were identified enriched for genes that responded to feeding by the sugar beet root maggot (SBRM) in both the moderately resistant genotype F1016 and the susceptible F1010 using SSH (Puthoff and Smigocki 2007). Of the examined ESTs, 20% were regulated by methyl jasmonate, 17% by salicylic acid and 11% by ethylene, suggesting that these signaling pathways were involved in sugar beet root defense response. Identification of these sugar beet root ESTs provided knowledge concerning plant root defense and would likely lead to the development of novel strategies for the control of the sugar beet root maggot. This research provided significant knowledge about how sugar beet protect itself against insect invasions.

Interestingly, there is a complementary transcriptome analysis of sugar beet root maggot (SBRM) (Tetanops myopaeformis) genes modulated by the infestation of sugar beet roots (Li and Smigocki 2018). SRBM, the most destructive insect pest of Beta vulgaris host, is the larva of a small fly, affecting more than half of all North American sugar beet acreage. SSH generated more than 300 SBRM ESTs differentially expressed in the interaction of the pest with a moderately resistant (F1016) and a susceptible (F1010) sugar beet line. Gene Ontology (GO) analysis predicted a dominance of metabolic and catalytic genes involved in the interaction of SBRM with its host. The functions of SBRM genes during development, regulation, cellular process, signaling and under stress conditions were annotated. This research provided the information on how insects evolve adaptive mechanisms to overcome host sugar beet resistance and develop tolerance to many insecticides used for their control.

10.4.4 The Tolerance and Adaptation Mechanisms in Transcriptomic Changes of Sugar Beet in Response to Biotic and Abiotic Stresses

More than 10,000 unigenes were differentially expressed under salt stress, 1205 unigenes were differentially expressed under cold stress, 150 ESTs from sugar beet and 300 ESTs from SBRM were profiled, which revealed the molecular mechanism of sugar beet in response to biotic and abiotic stresses in transcriptomic changes.

10.5 The Proteome Analysis of Sugar Beet in Response to Biotic and Abiotic Stresses

Because of post-transcriptional events and post-translational modifications such as phosphorylation and glycosylation, it is of essential importance to study stress responses at the protein level. As a necessary and complementary approach in the postgenomic era, proteome analysis is a relatively new and a powerful set of tools for studying large-scale plant responses to biotic and abiotic stresses (Pandey and Mann 2000; Wakeel et al. 2011b). Recent analyses of proteomes in sugar beet in response to salt stress and other stresses have yielded more information for understanding the complex mechanisms of sugar beet stress response and tolerance (Table 10.1).

Table 10.1 The proteomic analysis of sugar beet under stress conditions (Summary of publications on sugar beet stress responsive proteomics)
Full size table

10.5.1 The Proteome Analysis of Sugar Beet in Response to Salt Stress

The changes in protein expression caused by salinity were monitored using two-dimensional gel-electrophoresis (2-DE) (Wakeel et al. 2011b). Most of the detected proteins in sugar beet showed stability under salt stress. The statistical analysis of detected proteins showed that the expression of only six proteins from shoots and three proteins from roots was significantly altered. At this stage, the significantly changed proteins expression detected could not be attributed to sugar beet adaptation under salt stress. However, unchanged membrane bound proteins under salt stress did reveal the constitutive adaptation of sugar beet to salt stress at the plasma membrane level.

The salt-tolerant mechanism of a special salt-tolerant germplasm resource, sugar beet M14, was studied in depth through a series of proteomic experiments. Sugar beet monosomic addition line M14 displayed interesting phenotypes such as apomixis and salt stress tolerance (Yang et al. 2012; Li et al. 2009). The M14 seedlings were grown in hydroponics with different NaCl concentrations (0 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM) (Yang et al. 2012). Morphological changes were observed 7 days after salt treatment (Yang et al. 2012). The M14 seedlings could survive 7 days of 500 mM NaCl treatment. Some M14 seedlings died at concentrations over 500 mM NaCl, and all of the M14 died at 900 mM NaCl. Therefore, 500 mM NaCl was a high limit concentration that M14 can survive, and it was used in this study to investigate high salt responsive proteins. The proteomic analysis of M14 leaves and roots under 500 mM NaCl treatment for 7 days were reported (Yang et al. 2012). Proteins from control and treated samples were extracted and separated using 2-DE. A total of 40 protein spots from leaf gels and 36 protein spots from root gels exhibited significant changes. Using mass spectrometry and database searching, 38 unique proteins in leaves and 29 unique proteins in roots were identified. The proteins included those involved in metabolism, protein folding, photosynthesis, and protein degradation. This research has revealed candidate proteins for detailed functional characterization, and set the stage for further investigation of the high concentration salt tolerance mechanisms in sugar beet M14. The differentially expressed proteins such as SAMS, glutamine synthetase (GS), carbamoyl-phosphate synthase (CPS), betaine aldehyde dehydrogenase (BADH), ferritin, heat shock proteins (HSPs), filamentation temperature-sensitive H (FtsH), proteasome proteins, glutathione S-transferase (GST) and 14-3-3s have provided interesting candidates for detailed characterization of their functions in salt stress tolerance. This study provided the mechanism of salt tolerance in sugar beet M14 under long-term (7 days) and high salt stress (500 mM NaCl) at the protein level.

In order to understand the tolerance mechanisms of sugar beet M14 under moderate salt stress (200 mM, 400 mM NaCl), the salt-responsive characteristics of the M14 plants under 0, 200, and 400 mM NaCl conditions using quantitative proteomics approaches were reported (Yang et al. 2013). Combining two-dimensional difference gel electrophoresis (2D-DIGE) and isobaric tag for relative and absolute quantification (iTRAQ) technologies, 142 differentially expressed proteins in leaves and 65 differentially expressed proteins in roots were identified under salt stress (Yang et al. 2013). The proteins were mainly involved in photosynthesis, energy metabolism, protein folding and degradation, and stress and defense. This study provided the mechanism of salt tolerance in sugar beet M14 under long-term (7 days) and moderate salt stress (200 mM, 400 mM NaCl) at the protein level, including enhancement of photosynthesis and energy metabolism, accumulation of osmolyte and antioxidant enzymes, and regulation of methionine metabolism and ion uptake/excretion.

Plant membrane proteins are known to play critical roles in salt stress signaling and adaptation (Gilmore and Washburn 2010; Vertommen et al. 2011). In order to identify significantly changed membrane proteins and determine their possible relevance to salt tolerance, the changes in membrane proteome of the M14 plants leaves in response to salt stress (0, 200, 400 mM NaCl) using an iTRAQ LC-MS/MS technology for quantitative proteomic analysis were reported (Li et al. 2015). Fifty membrane proteins exhibiting differential protein level changes were identified, which were mainly involved in transport, metabolism, protein synthesis, photosynthesis, protein folding and degradation, signal transduction, stress and defense, energy metabolism, and cell structure. This study provided the membrane adaptive mechanisms in sugar beet M14 under long-term (7 days) and moderate salt stress (200 mM, 400 mM NaCl) at the protein level.

Protein phosphorylation is one of the most common and important post-translational modifications (PTMs), which plays important roles in the regulation of diverse processes including metabolism, transcription/translation, protein degradation, homeostasis, cellular communication/signaling, proliferation, differentiation, and cell survival (Hunter 2000; Thingholm et al. 2009). Plants respond to salt stress by triggering phosphorylation cascades (Gong et al. 2004). In order to identify significantly changed proteins and phosphoproteins and determine their potential relevance to salt stress response, the characteristics of the M14 plants leaves at time points 30 min and 60 min after 0, 200, and 400 mM NaCl treatment using label-free quantitative proteomics approaches were reported (Yu et al. 2016). One hundred fourteen differentially expressed proteins were identified under salt stress. One hundred eighty nine phosphoproteins exhibited significant changes at the phosphorylation level under salt stress. The salt stress responsive proteins and phosphoproteins were mainly involved in signal transduction and kinase, transport, photosynthesis, metabolism, protein folding and degradation, and stress and defense. Exciting findings from this quantitative phosphoproteomics study include: (1) Several signaling components kinases under short term salt stress were found, e.g. 14-3-3 and mitogen-activated protein kinases (MAPK) and calcium-dependent protein kinases (CDPK); (2) Protein phosphorylation events were not limited to signal transduction spreading across key physiological processes including transport, transcription, metabolism, and stress and defense. This explained how the M14 plants can quickly respond to salt stress, make adaptive changes in cellular biochemical and physiological processes, and achieve tolerance for long-term growth and development. This study revealed the salt responsive mechanisms of the special sugar beet M14 line under short-term (30 min and 60 min) and moderate salt stress (200 mM, 400 mM NaCl) using label-free quantitative phosphoproteomics.

Acetylation of histone proteins is a type of chromatin modification that facilitates the activation of genes in plant defense against a variety of environmental stresses (Kim et al. 2015; Hu et al. 2011). Deciphering the exact mechanisms of chromatin modifications under abiotic stress conditions is important for improving crop plants’ performance and yield. The salt stress responses of B. v. vulgaris and B. v. maritima were compared (Yolcu et al. 2016). The chromatin immunoprecipitation (ChIP) assay in control and salt stressed (250 and 500 mM NaCl) plants were performed and the enrichment of acetylation in the associated chromatin sites was compared. The transcriptional activation of one POD encoding gene that was associated with the elevated levels of acetylation in H3K9 and H3K27 sites was found. The acetylation patterns were remarkably different between two subspecies in which the highest acetylation levels were found at H3K9 and H3K27 in wild beet and sugar beet, respectively.

10.5.2 The Proteome Analysis of Sugar Beet in Response to Other Stresses

To investigate how the sugar beet was affected by K+ deficiency and by substitution of K+ by Na+, the comparative proteomic approach 2-DE was applied to obtain 27 significant changes protein among the treatments (Pi et al. 2016). Based on the proteomic results, K+ was assumed to engage in a wide range of metabolic pathways, especially photosynthesis and cellular respiration. It was found that Na+ may stimulate photosynthesis and restrict cellular respiration in K+ deficiency. Na+ was therefore in some ways able to recover damage due to K+ deficiency at the protein level, but cannot functionally replace K+ as an essential nutrient.

Iron is the fourth most abundant element in the Earth’s crust and is an essential micronutrient for all living organisms including plants. However, its low availability in neutral or alkaline soils, which account for approximately 30% of the world’s arable soils, causes Fe deficiency (Abadía et al. 2004). Iron deficiency is the most common nutritional disorder in many plants and typical symptoms include chlorosis of young leaves (leaf yellowing) and reduced crop yield and quality (Abadía et al. 2011). In order to understand the Fe deficiency mechanisms of sugar beet, the protein profile changes of the sugar beet apoplastic fluid induced by Fe deficiency and Fe resupply to Fe-deficient plants were studied by 2-DE (Ceballos-Laita et al. 2015). One hundred nine unique proteins were identified, 75% of which were classified into stress and defense, protein metabolism, cell wall and carbon metabolism. The result showed that protein homeostasis in the leaf apoplast fluid was well-maintained upon Fe shortage. The identification of three chitinase isoforms among proteins increasing in relative abundance with Fe-deficiency suggested that one of the few effects of Fe deficiency in the leaf apoplast proteome included cell wall modifications. Iron resupply to Fe deficient plants changed the relative abundance of 16 spots when compared to either Fe-sufficient or Fe-deficient samples.

Plants grown under iron deficiency show different morphological, biochemical and physiological changes (Santi and Schmidt 2009; Jin et al. 2007). These changes include the elicitation of different strategies to improve the acquisition of Fe from the rhizosphere, the adjustment of Fe homeostasis processes and a reorganization of carbohydrate metabolism (Larbi et al. 2010; López-Millán et al. 2001). In order to provide a holistic view of the metabolic processes occurring in sugar beet plants under different Fe status, the changes induced in the root tip proteome and metabolome of sugar beet plants in response to Fe deficiency and resupply were characterized (Rellán-Alvarez et al. 2010). By 2-DE, iron deficiency resulted in changes in the relative amounts of 22 proteins. Metabolites in root tip extracts were analyzed by gas chromatography-MS, 26 identified metabolites changed significantly with Fe deficiency. Iron deficiency induced increases in the relative amounts of proteins and metabolites associated to glycolysis, tricarboxylic acid cycle (TCA) and anaerobic respiration. Furthermore, a protein not present in Fe-sufficient roots, dimethyl-8-ribityllumazine (DMRL) synthase, was present in high amounts in root tips from Fe-deficient sugar beet plants and gene transcript levels were higher in Fe-deficient root tips. The increases in DMRL synthase and in raffinose family of oligosaccharides (RFO) sugars were the major changes induced by Fe deficiency and resupply in root tips of sugar beet plants. This study confirmed the increase in proteins and metabolites related to carbohydrate metabolism and TCA cycle pathways.

Zinc is an essential micronutrient for plants and plays structural and/or catalytic roles in many essential physiological processes, being the only metal present in all enzyme classes and the second most abundant transition metal after Fe (Vallee and Auld 1990). However, when present at high concentrations, Zn can be toxic to plants (Foy et al. 1978; Singh and Agrawal 2007). To investigate comprehensively the effects of Zn toxicity in roots of sugar beet plants, changes induced by three levels of Zn in the root proteome from B. vulgaris were studied by 2-DE (Gutierrez-Carbonell et al. 2013). Three hundred twenty spots were consistently detected and 16, 16 and 35 of them showed significant changes in relative abundance as a result of the 50, 100 and 300 μM Zn treatments, respectively, when compared to the control (1.2 μM Zn). Forty-four spots had consistent changes between all treatments, and 41 differentially expressed proteins were identified. At low and mild Zn excess, Complex I of the mitochondrial transport chain and the oxidative phosphorylation were mildly impaired, and an effort to compensate this effect by increasing glycolysis was observed. At high Zn excess, a general metabolism shutdown occurred, as denoted by decreases in the aerobic respiration and by an impairment of the defense systems against oxidative stress. This study suggested that metabolic changes at high Zn supply reflected cell death, while changes at low and mild Zn supplies may rather explain the metabolic reprogramming occurring upon Zn toxicity. Results also suggested that Zn competition with divalent ions including Fe may contribute to many Zn toxicity symptoms, especially at low and moderate Zn supplies.

10.5.3 The Tolerance and Adaptation Mechanisms in Translational Level Changes of Sugar Beet in Response to Biotic and Abiotic Stresses

Proteomics research in sugar beet was mainly focused on salt stress. Four hundred thirty eight differentially expressed proteins in sugar beet M14 involved in short-term and long-term salt stress responses and 9 additional differential expression proteins from Beta vulgaris L. cv. Evita under salt stress were identified and classified into different functions. Interestingly, 189 differentially expressed phosphoproteins at the phosphorylation level and two acetylated proteins at acetylation levels were found in sugar beet in response to salt stress. One hundred ninety nine differentially expressed proteins in sugar beet which participated in other abiotic stresses were identified and analyzed. Proteomics research in sugar beet provides a large number of stress-responsive proteins resources, which has a prospect to improve stress resistance of sugar beet and other crops by genetic engineering.

10.6 Conclusion

In this paper, we have reviewed the mechanisms of sugar beet responses to biotic and abiotic stresses at the physiological, gene functional, transcriptional and translational levels. For physiological changes, most research has been carried out for salt and drought stresses. It was concluded that the increase in the content of enzymes and non-enzymatic antioxidants were also the main strategies for sugar beet to regulate ROS and redox homeostasis under salt, drought and various stresses. The ability of keeping Na+/K+ ratio stable and cytosolic K+ homeostasis were the main factors for sugar beet in response to salt stress. The increase in proline is one of the main physiological indicators for sugar beet in response to drought stress. The functions of genes from sugar beet in response to salt, cold and heavy metal stresses were mainly investigated by transgenic technologies. BvPGDHa, BvSHMTa, BvCMO, BvNHX1, BvM14-glyoxalase I, BvM14-cystatin and BvM14-SAMS2 conferred salt tolerance for sugar beet; the transcriptional level of BvpAPX and the gene families of BvSOD, BvPrX, BvAPX, BvPTOX and BvRBOH were induced by salt stress. BvIMP was involved in cold tolerance for sugar beet. Transgenic sugar beets carrying the StGCS-GS gene from Streptococcus thermophiles accumulated more Cd, Zn and Cu ions in shoots than wild-type. The roles of StGCS-GS in enhancing Cd, Zn and Cu tolerance and accumulation in transgenic sugar beet may represent a highly promising new tool for phytoremediation. At the transcriptional level, the transcriptome analysis of sugar beet in response to salt, cold and biotic stresses were conducted by RNAseq or SSH methods. At the level of translation, more than 800 differentially expressed proteins in response to salt, K+/Na+ ratio, Fe deficiency, Fe resupply and heavy metal Zn conditions were identified by quantitative proteomics techniques, which were mainly involved in multiple metabolic pathways including photosynthesis, metabolism, protein folding and degradation, transport, stress and defense and other pathways. These studies have provided new insights unveiling the mechanisms of sugar beet in response to biotic and abiotic stresses.