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Sugar beet root susceptibility to
storage rots and downregulation of
plant defense genes increases with
time in storage
Shyam L. Kandel1, John D. Eide1, Andrea Firrincieli2, Fernando L. Finger3, Abbas M. Lafta4
& Karen K. Fugate1
Storage rots are a signicant cause of postharvest losses for the sugar beet crop, however, intrinsic
physiological and genetic factors that determine the susceptibility of roots to pathogen infection
and disease development are unknown. Research, therefore, was carried out to evaluate the disease
development in sugar beet roots caused by two common storage pathogens as a function of storage
duration and storage temperature, and to identify changes in the expression of defense genes that
may be inuencing the root susceptibility to disease. To evaluate root susceptibility to disease, freshly
harvested roots were inoculated with Botrytis cinerea or Penicillium vulpinum on the day of harvest
or after 12, 40, or 120 d storage at 5 or 12 °C and the weight of rotted tissue present in the roots after
incubation for 35 d after inoculation were determined. Disease susceptibility and progression to B.
cinerea and P. vulpinum increased with storage duration with elevations in susceptibility occurring
more rapidly to B. cinerea than P. vulpinum. Also, B. cinerea was more aggressive than P. vulpinum
and caused greater rotting and tissue damage in postharvest sugar beet roots. Storage temperature
had minimal eect on root susceptibility to these rot-causing pathogens. Changes in defense gene
expression were determined by sequencing mRNA isolated from uninoculated roots that were similarly
stored for 12, 40 or 120 d at 5 or 12 °C. As susceptibility to rot increased during storage, concurrent
changes in defense-related gene expression were identied, including the dierential expression of 425
pathogen receptor and 275 phytohormone signal transduction pathway-related genes. Furthermore,
plant resistance and hormonal signaling genes that were signicantly altered in expression coincident
with the change in root susceptibility to storage rots were identied. Further investigation into the
function of these genes may ultimately elucidate methods by which storage rot resistance in sugar
beet roots may be improved in the future.
Keywords Botrytis, Pathogen receptor, Phytohormone signaling, Penicillium, Storage, Sugar beet
Sugar beet (Beta vulgaris L.) taproots contain up to 21% sucrose by fresh weight and are used to manufacture
sugar on an industrial scale1,2. e Red River Valley (RRV) of Minnesota and North Dakota leads the United
States in sugar beet production, contributing nearly 58% of total domestic production. Due to high tonnage of
the crop that exceeds immediate sugar factory processing capabilities, postharvest sugar beet roots are oen
stored for many weeks before processing. In the RRV of Minnesota and North Dakota, sugar beet roots are stored
in large outdoor piles or ventilated sheds for up to 280 d prior to processing3. During storage, it is estimated
that respiration causes 50–80% sucrose loss4–6. Furthermore, elevated temperature and free moisture inside the
storage piles promote microbial activities leading to the rapid deterioration and soening of root tissues7,8. Also,
sugar beet roots are prone to mechanical damage during leaf removal, harvesting, and loading and reloading of
roots during transportation and storage. Damaged areas of the root are susceptible to disease since they provide
microbes access to internal root tissue.
1Edward T. Schafer Agricultural Research Center, Sugarbeet Research Unit, USDA-ARS, Fargo, ND 58102,
USA. 2Department for Innovation in Biological, Agro-Food and Forest Systems, University of Tuscia,
Viterbo, Italy. 3Departamento de Agronomia, Universidade Federal de Vicosa, 36570-900 Vicosa, MG, Brazil.
4Department of Plant Pathology, North Dakota State University, P.O. Box 6050, Fargo, ND 58108, USA. email:
Shyam.Kandel@usda.gov
OPEN
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Several fungi and bacteria can cause storage rots or postharvest diseases and sugar loss in sugar beet in the
U.S. and elsewhere7,9–14. Botrytis cinerea and Penicillium spp. are reported as major fungal pathogens associated
with storage rots in sugar beet7. B. cinerea and Penicillium species are necrotrophic fungi which can infect and
cause postharvest losses in many agricultural crops including sugar beet11,14–16. Both pathogens require exposed
tissues (e.g., wounds, cracks, or bruises) for infection and colonization of root tissue. Cell wall degrading enzymes
and toxins secreted by Botrytis and Penicillium pathogens facilitate the initial stage of infection and subsequent
colonization17–19. Following colonization, infected tissue collapses and rotted, moldy disease symptoms occur
which can be easily noticed by the naked eye. In general, abundant fungal biomass and spore masses are produced
in the decaying area of infected sites. Plant defense mechanisms utilized by sugar beet roots to limit storage rots
and the factors aecting these mechanisms still need to be determined.
Presently, host resistance in sugar beet cultivars to limit storage diseases and sucrose losses has been largely
unexplored due to a lack of knowledge regarding the genetic and molecular basis of defense responses during the
storage. Host resistance has been utilized to minimize the damage caused by Cercospora leaf spot or Rhizomania
disease in sugar beet20,21, but no genes that regulate or contribute to immune responses or pathogen resistance
in postharvest sugar beet roots have been identied. In plants, pathogen receptor proteins and phytohormones
play signicant roles in the provision of protection from abiotic and biotic stresses22–25 and are likely to inuence
disease development in stored sugar beet roots. RNA sequencing (RNA-seq) technology has enabled study of the
genome-wide expression of stress and disease responsive genes in a variety of crop plants26–30. In this study, we
determined the eect of storage time and temperature on the severity of postharvest rots caused by the common
storage pathogens; B. cinerea and P. vulpinum. We also identied pathogen receptor and phytohormone signal
transduction genes that were altered in expression with respect to storage with a potential role in host resistance.
To the best of our knowledge, this is the rst report of genome-wide transcriptional changes in defense-related
genes that are altered in expression during storage of sugar beet roots.
Materials and methods
Plant materials, storing of sugar beet roots, pathogen inoculation, and disease assessment
Sugar beets (variety VDH66156, SESVanderHave, Tienen, Belgium) were grown in a greenhouse in 15L pots with
16h light/8h dark periods as previously described31. Roots were harvested from 16 to 17 weeks old plants. All
leaf material was removed, and roots were washed gently to remove adhering potting mix. On the day of harvest
(0 d), eight randomly selected roots were inoculated with Botrytis cinerea, and an additional eight randomly
selected roots were inoculated with Penicillium vulpinum (formerly P. claviforme32) to evaluate susceptibility
to storage rot from these two pathogens using assays described below. Pathogen cultures were obtained from
diseased roots collected from commercial sugar beet piles by W. Bugbee (USDA-ARS, Fargo, ND, retired). e
remaining roots were randomly divided into two groups. One group was stored at 5°C and the other group
was stored at 12°C, with roots at both temperatures stored at 95% relative humidity. Aer 12 d in storage, eight
random roots from each storage temperature were inoculated with B. cinerea and an additional eight random
roots per storage temperature were inoculated with P. vulpinum for rot susceptibility assays. Similar operations
of roots and inoculations were carried out aer 40 and 120 d using roots stored at both 5 and 12°C.
Roots were inoculated and assessed for their susceptibility to storage rot from B. cinerea or P. vulpinum using
the protocol of Fugate et al.33. Two holes, 12 × 10mm (diameter x depth), were drilled on opposing sides of each
taproot where root girth was greatest. A mycelia-covered agar plug (10mm diameter), cultured as previously
described33, was inserted into each hole, with mycelia facing the exposed root tissue at the base of the hole.
Following inoculation, roots were incubated in a 20°C, 95% relative humidity growth chamber (Conviron,
model PGR15). Aer ve weeks incubation, susceptibility to rot was evaluated by excising and weighing the
rotted tissue from each inoculation site. Weight of the excised tissue from the two inoculation sites per root were
averaged to generate a single value for each root. e experiment was conducted with eight replications for each
storage temperature x storage time combination, with individual roots as the experimental unit. Roots inoculated
with B. cinerea were evaluated independently from roots inoculated with P. vulpinum, and the experiment was
repeated three times. e analysis of variance (ANOVA) with Fisher’s LSD was used to identify temperature and
storage duration treatments that diered signicantly.
Storage experiments and sample collections
In a separate experiment, taproots were harvested from 42 plants, and used to evaluate storage-related
transcriptional changes in sugar beet roots. Tissue samples were collected individually from six roots on the
day of harvest. Roots were longitudinally sectioned into four quarters and the tissue from one quarter section
of each root, which was representative of the entire root (root crown to root tail) including epidermal tissue and
the central vascular cylinder was collected. Tissue samples were rapidly frozen in liquid nitrogen, lyophilized,
ground to a ne powder, and stored at -80°C prior to use. e remaining roots were randomly divided into two
groups which were stored in the two chambers of a Conviron (Winnipeg, MB, Canada) E7/2 two-tier growth
chamber, with one chamber operating at 5°C, the other operating at 12°C, and both chambers set to 95%
relative humidity. Six roots were randomly removed from each chamber aer 12, 40, and 120 d in storage and
tissue samples were collected from these roots as described above. e experiment was conducted with six
replications for each storage temperature x storage time combination, with individual roots as the experimental
unit. Sugarbeet plants used in our experiments were grown in the greenhouse as per USDA-ARS guidelines and
comply with relevant institutional, national, and international guidelines and legislation.
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RNA isolation, sequencing, and data analysis
Total RNA was extracted from lyophilized sugar beet root tissue using a RNeasy Plant Mini Kit (QIAGEN,
Valencia, CA) with an on-column DNase digestion. An entire root section was pulverized to a very ne powder,
mixed well, and 50mg of pulverized powder was used for the RNA extraction. RNA concentration was quantied
using a ermoFisher Scientic NanoDrop ND-1000 (Waltham, MA) and RNA integrity was conrmed using
an Agilent Technologies 2100 Bioanalyzer (Pal Alto, CA). RNA was fragmented, converted to cDNA using
random primers, amplied by PCR, and sequenced by BGI Americas (Cambridge, MA) using a BGISEQ-500
sequencing system, DNA nanoball technology, and 100bp paired-end sequencing. An average of 29.3M raw
reads was generated per sample. Sequencing reads were processed using SOAPnuke ver. 1.5.234 with > 28million
high-quality (HQ) reads per sample. HQ reads were mapped to the sugar beet genome35 using Bowtie2 ver.
2.2.536 and gene expression levels were calculated with RSEM version 1.2.1237. Dierentially expressed genes
(adjusted-p-value < 0.01 and absolute log2 fold change > 1.0) were detected using DEseq238.
Identication of dierentially expressed plant resistance and phytohormone related
genes
e Pathogen Receptor Genes database (PRGdb: http://prgdb.org/prgdb4/)39 was used to identify pathogen
receptor genes in sugar beet that were dierentially expressed during storage (5or 12°C for 12, 40, or 120
d versus 0 d). Specically, B. vulgaris aliases (BVRB_*) identied as pathogen receptor genes were manually
downloaded from PRGdb and used to recover the corresponding Entrez gene ID from the National Center for
Biotechnology Information (NCBI) database39,40. PCA was performed to display the direction of variability in
dierential expression of pathogen receptor genes and visualized using the factoextra R package41.
e DEGs involved in the phytohormone signal transduction pathways were recovered by using the
functional annotation based on KEGG database and identied KEGG orthologs involved in ABA, auxin, BR,
CTK, ET, GA, JA, and SA signaling pathways42,43.
Upset plots and heatmaps for signicantly dierentially expressed (up-and down-regulated) genes were
generated using the ComplexHeatmap R package44.
Results
Storage duration and temperature eects on disease susceptibility
B. cinerea and P. vulpinum infected and caused rotting symptoms in sugar beet roots that were inoculated with
these pathogens at 0 d or aer storage for 12, 40, and 120 d at 5or12 °C (Figs.1 and 2). Both pathogens
caused signicantly greater rotting symptoms in roots that were stored for 40 or 120 d compared to 0 or 12 d,
indicating that root defenses against the fungal pathogens diminished with storage duration. Aer 40 d storage
at 12°C, B. cinerea inoculated roots had signicantly more rotted tissue compared to roots inoculated on 0, 12
or 120 d at 5 and 12°C post storage (Fig.2). In P. vulpinum inoculated roots, signicantly more rotted tissue
was observed at 40 and 120 d post storage than at 0 and 12 d, and the maximum weight of rotted tissues was
observed in roots that were inoculated aer storage for 120 d at 12°C. Rotted lesions were generally larger in
roots inoculated with B. cinerea than roots inoculated with P. vulpinum (Fig.1). Furthermore, both pathogens
caused the characteristic blackish-brownish discoloration in the internal root tissues at inoculation sites (Fig.1).
In general, storage temperature had minimal eect on the susceptibility of roots to storage rot caused by either
pathogen. No signicant dierence in the weight of damaged root tissue was found between roots stored at 5
and 12°C for either pathogen except for B. cinerea-infected roots inoculated aer 40 d or P. vulpinum-inoculated
roots aer 120 d (Fig.2). Results of disease assays indicate that susceptibility to B. cinerea and P. vulpinum
increases with storage duration, with rot symptoms of greater severity with B. cinerea than P. vulpinum, but
minimal at higher temperature.
Storage duration and temperature eects on expression of plant pathogen receptor
genes
Dierentially expressed pathogen receptor genes were identied by RNA sequencing of sugar beet roots that
were freshly harvested (0day) and stored for 12, 40 or 120 d at 5 and 12°C. A total of 425 pathogen receptor genes
were found dierentially expressed in stored sugar beet roots (Supplementary File 1). A principal component
analysis (PCA) of dierentially expressed genes (DEGs) indicates a clear dierence in the expression of pathogen
receptors in stored versus freshly harvested roots (Fig.3). e storage time-dependent clustering highlights a
shi in the expression of pathogen receptor genes in stored roots at 12, 40, and 120 d (Fig.3). At 12 and 40 d,
distinct clustering of these genes between two storage temperatures, 5 and 12°C was observed, but no such
dierence was found at 120 d (Fig.3). A total of 75 and 149 pathogen receptor genes were signicantly up-
and down-regulated (absolute log2fold change > 2.0 and P-adj < 0.01), respectively, in stored sugar beet roots
(Fig.4A-B). Nearly 75% of signicantly expressed receptor genes were down-regulated as a general response to
storage (Fig.4), with 22 genes down-regulated across all storage treatments (Fig.4B). On the other hand, three
genes were up-regulated across all storage treatments (Fig.4A). More pathogen receptor genes were down-
regulated at 40 d compared to other storage times.
Among down regulated pathogen receptor genes, 39 genes with protein kinase domains and 33 genes with
receptor-like protein kinase domains were down-regulated during storage (Figs.5 and 6). Nine genes with a
protein kinase or receptor-like protein kinase domain were down-regulated in all storage conditions. Genes
related to leucine or probable leucine rich receptor or receptor-like kinases, wall-associated receptor kinases,
and serine/threonine-protein kinases were major receptor genes signicantly down-regulated during storage
(Figs.5 and 6). Fewer receptor genes containing protein kinase or receptor-like protein kinase domains were
signicantly up-regulated in response to storage (Fig.7). Genes related to mitogen-activated protein kinase 4
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(MAPK4) and serine/threonine-protein kinase AtPK2/AtPK19 were signicantly up-regulated across storage
treatments (Fig.5). Other infrequent receptor genes with coiled-coil or lectin kinase domain were variably up or
down-regulated during storage (Supplementary Fig.1).
Storage duration and temperature eects on expression of phytohormone signaling
genes
A total of 275 dierentially expressed genes involved in abscisic acid (ABA), auxin, brassinosteroid (BR),
cytokinin (CTK), ethylene (ET), gibberellic acid (GA), jasmonic acid (JA), and salicylic acid (SA) signal
transduction pathways were identied using the KEGG plant hormone database42,43. A total of 72 and 93
phytohormone signaling pathway genes were signicantly up-and down-regulated (absolute log2fold change > 2.0
and P-adj < 0.01), respectively. e expression of phytohormone signaling genes was aected by both storage
duration and temperature. However, storage time rather than storage temperature had a larger inuence on
the dierential expression of signaling genes. In total, up-regulated genes increased their expression from 12 to
40 to 120 d of storage (Fig.8A), while down-regulated genes increased from 12 to 40 d but remained similar at
40 and 120 d (Fig.8B). Dierentially expressed genes involved in ethylene, JA, and SA were examined further
due to the well-dened role of these hormones in plant defense responses (Fig.9)22. Multiple genes encoding
ethylene-insensitive protein 2 (ethylene signaling pathway), transcription factor MYC2 (JA signaling pathway),
and transcription factor TGA (TGACG MOTIF-BINDING FACTOR) (SA signaling pathway) were signicantly
altered in expression during storage. Some genes related to ethylene-responsive transcription factor 1 (ethylene
signaling pathway), transcription factor MYC2 (JA signaling pathway), and pathogenesis-related protein 1 (PR-
1) and transcription factor TGA (SA signaling pathway) were signicantly down-regulated at one or more time
points (Fig.9). Several genes related to ABA, auxin, BR, CTK, and GA signaling pathways were also up-or down-
regulated (Supplementary Figs.2,3) in stored roots and are listed in the Supplementary File 2.
Fig. 1. Rot symptoms on sugar beet roots caused by (A) Botrytis cinerea and (C) Penicillium vulpinum.
Longitudinal sections of sugar beet roots displaying internal rot symptoms of B. cinerea (B) and P. vulpinum
(D). Sugar beet roots were stored for 0, 12, 40, and 120 d at 5or12°C, aer which they were inoculated and
incubated for 35 d at 20°C and 95% relative humidity.
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Discussion
Storage diseases can cause signicant sucrose loss and quality deterioration in postharvest sugar beet roots.
Botrytis cinerea and P. vulpinum are major pathogens of stored sugar beet roots7,8,14. Understanding the biology
of storage diseases and defense responses is vital to minimize sugar loss from postharvest sugar beet roots. In this
study, eects of storage duration and storage temperature on disease susceptibility were assessed by inoculating
roots with B. cinerea and P. vulpinum at dierent storage times. Additionally, changes in the expression of defense
related genes were identied in sugar beet roots that may inuence changes in innate immunity or susceptibility
to these pathogens.
Botrytis cinerea is a necrotrophic pathogen which causes gray mold diseases and can infect more than 200
plant species, including sugar beet roots in storage16,45. In the process of suppressing host defenses and initiating
the infection, this fungus secretes cell wall degrading enzymes and reactive oxygen species that degrade and kill
host cells16,17. In our study, sugar beet roots inoculated with B. cinerea were found most susceptible at 40 d post
storage compared to 0, 12 or 120 d (Fig.2A) indicating that storage time can aect the susceptibility of roots
to disease development and root deterioration. e underlying mechanism causing the higher tissue damage at
Fig. 2. Fresh weight of rotted tissue in sugar beet roots inoculated with (A) Botrytis cinerea and (B) Penicillium
vulpinum. e weight of rotted tissue was measured at 35 d aer pathogen inoculation. Means that do not
share a letter are signicantly dierent (P < 0.05) according to Fisher’s Least Signicant Dierence (LSD)
Method. Vertical error bars represent standard error of means.
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40 d is not known, although it is possible that the progressive cell wall changes occurring during storage could
aect disease susceptibility and lesion spreading. Furthermore, at 40 d, signicantly more root deterioration
was observed at 12°C as compared to 5°C indicating that root susceptibility likely increases with an increase in
storage temperature. Like B. cinerea, P. vulpinum is a polyphagous necrotrophic pathogen which can infect many
postharvest crop commodities including sugar beet7,14. In sugar beet, P. vulpinum has commonly been isolated
from infected roots in storage and is associated with root rotting and necrotic lesions with blue mold symptoms.
In our disease assays, sugar beet roots stored for 40 and 120 d and subsequently inoculated with P. vulpinum
showed signicantly higher rot symptoms and tissue damage than 0 or 12 d of storage (Fig.2B). e eect of
temperature on the severity of rotting was only signicant in roots stored for 120 d where higher rotted tissue
was observed at 12°C than 5°C (Fig.2B). Past studies were performed to understand physiological processes
and enzymatic activities that lead to the sucrose losses in stored roots5,46, while this study provides new insights
into how storage time and temperature aects the susceptibility or defensive capability of sugar beet roots against
microbial pathogens. Our results suggest that storage time and storage temperature are critical determinants for
increasing susceptibility of sugar beet roots to storage pathogens such as B. cinerea and P. vulpinum. Interestingly,
Fig. 3. PCA plot of dierentially expressed pathogen receptor genes in sugar beet roots during storage.
e percentage of variation on the rst and second axes was calculated based on log2 transformed FPKM
expression data across treatments at 0, 12, 40, and 120 d of storage for roots stored at 5or12°C. e 95%
condence ellipses were inserted covering six replicates of each treatment.
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rotting damage in B. cinerea inoculated roots was twice that of P. vulpinum inoculated roots, especially at 40 and
120 d, although no such discrepancy was observed in roots inoculated at 0 d storage (Fig.2). Perhaps, B. cinerea
is more aggressive and rapidly overcomes the defensive capability of roots that were stored for longer times, such
as 40 and 120 d. Likewise, increased down-regulation of pathogen receptor genes at 40 and 120 d may imply
declining of innate immunity in stored roots, which corroborates with disease progression with storage time.
Despite prior work on the genetics of host-pathogen interactions for foliar or root diseases in sugar beet21,47,
little is known about defense-related transcriptional changes in sugar beet roots during storage. Pathogen receptor
and phytohormone signaling genes have a signicant role in plants to activate and confer defense responses
against challenges from microbial pathogens23–25. In this study, transcriptional changes of pathogen receptors
and phytohormone signaling genes during sugar beet storage were identied. Storage altered the expression of
pathogen receptor genes as the genes expressed in freshly harvested roots (0day) diered from stored roots
under variable temperatures and times (Fig. 3). Storage caused hundreds of pathogen receptor genes to be
dierentially expressed, with distinct transcriptional responses to storage temperature and time found. Sugar
beet roots undergo genetic and physiological changes during storage as observed in other crops which may lead
to the transcriptional reprograming of expression of plant resistance genes48,49. Transcriptional reprograming
began within a few days of storage as indicated by the large number of up- and down-regulated genes that were
identied already at 12 d storage. As storage continued, a maximum in the number of pathogen receptor genes
that were down-regulated occurred at 40 d which correlates with our ndings of increased root susceptibility
and maximum tissue damage. ese results are consistent with the idea that immunity is compromised in stored
sugar beet roots through the downregulation of resistance genes. Past studies also suggested that downregulation
of immune receptor genes increases host susceptibility and disease damage26,50,51. Receptor genes that were
down-regulated between 12 and 40 d are potential candidates for future research to understand the molecular
basis of diminishing host resistance during storage as this is the time when susceptibility increases the most.
On the other hand, some pathogen receptor genes were up-regulated in stored sugar beet roots which may
be crucial to maintain defense activities and maintain root health during storage (Fig.7). e upregulation of
genes encoding for MPK4 and serine/threonine-protein kinase AtPK2/AtPK19 potentially activates signaling
Fig. 4. UpSet plots of signicantly up (A) and down (B) regulated pathogen receptor genes (absolute log2fold
change > 2.0 and P-adj < 0.01) in stored sugar beet roots. Total counts of up-and down-regulated genes in each
treatment were displayed as horizontal bars, located on the le side of each panel. Vertical black lines with
lled dots indicate sets of intersections between two or more treatments. Vertical bars in the upper portion
of each panel represent total counts of unique or shared genes across treatments. e list of up-and-down
regulated genes was included in Supplementary File 2.
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pathways and cytoskeleton organization in sugar beet roots as documented in other plants to preserve root
health during storage52,53, but this awaits further validation.
Hormone signal transduction pathways are important in plant defense responses23,54 and many changes
in genes of ABA, auxin, BR, CTK, ET, GA, JA, and SA signaling pathways were also noted. Overall, more
phytohormone signaling genes were down-regulated versus up-regulated in stored sugar beet roots (Fig.8).
A similar pattern of down regulation of pathogen receptor and phytohormone signaling genes were identied,
suggesting there was direct or indirect co-regulation of these genes. Ethylene, JA, and SA signal transduction
genes such as transcription factor TGA, PR1 proteins, and transcription factor MYC2 were down-regulated
Fig. 5. Heat map of down-regulated receptor genes with protein kinase domains (KIN) in postharvest
sugar beet roots. e asterisk (*) indicates signicantly expressed genes (absolute log2fold change > 2.0 and
P-adj < 0.01). Squares are colored by dierential expression status. LFC = Log2fold change.
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which probably cooperated with down-regulated pathogen receptor genes to compromise the defensive capability
of sugar beet roots during storage. Nevertheless, the upregulation of hormonal signaling genes may have a
general eect on the ability of a root to defend itself during storage. Phytohormones such as ET, JA, and SA are
considered as key modulators to induce resistance and improve storage life of many postharvest products55. e
upregulation of ET, JA, and SA signal transduction genes can activate metabolic and signal transduction pathways
leading to increasing cell wall rigidity and antioxidant enzyme activities, delaying senescence, and defensive
gene expression during storage56–58. In sugar beet, postharvest jasmonate treatments stimulated accumulation of
antimicrobial and antioxidant compounds, improved mechanical strengths of cell walls, and provided resistance
to storage rots33,59. Future work on key regulators of mutual or exclusive interactions of pathogen receptor and
Fig. 6. Heat map of down-regulated receptor genes with receptor-like protein kinase domains (RLK) in
postharvest sugar beet roots. e asterisk (*) indicates signicantly expressed genes (absolute log2fold
change > 2.0 and P-adj < 0.01). Squares are colored by dierential expression status. LFC = Log2fold change.
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phytohormone signaling genes during storage will be useful to improve the host resistance for managing storage
diseases and minimize the sugar loss in sugar beet.
In this study, we demonstrated that the ability of roots to defend themselves against two major storage
pathogens declines in storage. Both storage temperature and duration aected the decline in the ability of roots
to defend themselves, although the eect of storage temperature was smaller than the eect of storage duration.
Large and complex changes in plant defense and hormonal signaling genes occurred in response to storage. Taken
Fig. 7. Heat map of upregulated receptor genes with protein kinase or receptor-like protein kinase domains
in postharvest sugar beet roots. e asterisk (*) indicates signicantly expressed genes (absolute log2fold
change > 2.0 and P-adj < 0.01). Squares are colored by dierential expression status. LFC = Log2fold change.
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together, this study provides an overview of defensive gene expression in sugar beet roots during storage, which
can guide additional studies to assess candidate genes for resistance to storage pathogens and understand how
the root’s innate defense system can be employed to improve storage. In future research, we will initiate studies
to understand the functional signicance of these genes to improve disease resistance and storage properties.
Fig. 8. UpSet plots of signicantly up (A) and down (B) regulated genes (absolute log2fold change > 2.0 and
P-adj < 0.01) involved in phytohormone signal transduction pathways in stored sugar beet roots. Total counts
of up-and down-regulated genes in each treatment were displayed as horizontal bars, located on the le side of
each panel. Vertical black lines with lled dots indicate sets of intersections between two or more treatments.
Vertical bars in the upper portion of each panel represent total counts of unique or shared genes across
treatments. e list of up-and-down regulated genes was included in Supplementary File 2.
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Fig. 9. Heat map of dierentially expressed genes involved in (A) ethylene, (B) jasmonic acid, and (C)
salicylic acid hormonal signal transduction pathways in postharvest sugar beet roots. e asterisk (*) indicates
signicantly expressed genes (absolute log2fold change > 2.0 and P-adj < 0.01). Squares are colored by
dierential expression status. K13413….K3449 are KEGG orthology identiers and indicate individual gene
functions on the pathways. Genes having the same molecular function is annotated with the same KEGG
identier. For example, genes 104899726and 104897480 were identied by KEGG as “Jasmonate ZIM-domain
containing protein” and, for this reason, annotated with the same KEGG Identier (aka KO) i.e. K13464.
Scientic Reports | (2024) 14:27235 12
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Data availability
e RNA-seq dataset generated in this study is available through the NCBI Sequence Read Archive under Bio-
Project identication number PRJNA938134.
Received: 22 May 2024; Accepted: 30 October 2024
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Author contributions
Conceived and designed the experiments: KKF and SLK. Performed the experiments: JDE, FLF, and AML. Ana-
lyzed the data: SLK, KKF, JDE, and AF. Contributed reagents/materials/analysis tools: SLK, KKF, and AF. Wrote
the paper: SLK, KKF, and AF.
Funding
Funding for this research was provided by the United States Department of Agriculture (USDA), Agricultural
Research Service through Project 3060-2100-045-00D. Mention of trade names or commercial products in this
publication is solely for the purpose of providing specic information and does not imply recommendation or
endorsement by the USDA. USDA is an equal opportunity provider and employer.
Declarations
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at h t t p s : / / d o i . o r g / 1
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