Introduction
Chinese cabbage (Brassica rapa ssp. pekinensis ) is the
most important leafy crop in Asia; the annual production of Chinese
cabbage in Asia is >50 million tons, which is approximately
70% of the world’s output (Su et al., 2018). Flowering or bolting time
is one of the most important traits in breeding Chinese cabbage because
premature flowering reduces, or completely damages in some severe cases,
the yield and quality of the harvested leafy products. With the
requirements for a year-round vegetable supply and effective use of
planting area, the demand for ecologically-targeted bolting-resistant
varieties is strong and is increasing (Su et al., 2018).
Decades of physiological studies have shown that flowering is initiated
in response to both environmental cues and endogenous pathways (Franks
& Weis, 2008). In Arabidopsis, flowering time is mainly regulated
through five pathways: vernalization, photoperiod, gibberellin,
autonomous, and aging (Fornara, de Montaigu, & Coupland, 2010). Several
abiotic stresses, such as drought and ambient temperature, can induce
flowering, suggesting that plants can integrate the effects of abiotic
stress with flowering signaling pathways (Andres & Coupland, 2012).
Chinese cabbage is a high water-consuming crop, and drought stress, a
very common abiotic stress that is caused by water deficit, causes a
series of physiological and molecular responses in plants. Water deficit
during the growing season or in some area can lead to early bolting and
insufficient vegetative growth, which ultimately affects the yield and
quality of Chinese cabbage. From a broad perspective, crop drought
sensitivity has been increased in the past two decades, and it is
predicted that drought stress will cause severe problems in plant growth
and crop production in more than 50% of agriculture lands by 2050
(Vurukonda, Vardharajula, Shrivastava, & Skz, 2015; Yang, Vanderbeld,
Wan, & Huang, 2010). Environmental adaptation is important for optimal
yield in the major crop plants. This includes adaptation of the
reproductive system to the prevailing climatic conditions and an
appropriate response to biotic and abiotic stresses. Flowering time is
an important trait with respect to drought adaptation, and it can
shorten the life cycle and lead to drought escape or avoidance (Araus,
Slafer, Reynolds, & Royo, 2002). In drought escape, plants flower
early, a strategy that allows the parent plant to produce seeds before
it is killed by drought (Fang & Xiong, 2014; Verslues & Juenger,
2011). However drought escape is fatal for leafy vegetables such as
Chinese cabbage. Therefore, breeding Chinese cabbage varieties with high
water use efficiency, which is simultaneously integrated with
bolting-resistant genetic components, is the best solution to this
issue.
In Arabidopsis, the flower-promoting gene GIGANTEA (GI ),
the florigen gene FLOWERING LOCUS T (FT ), andSUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1 ) play a
central role in the drought escape response (Blanvillain, Wei, Wei, Kim,
& Ow, 2011; Riboni, Galbiati, Tonelli, & Conti, 2013). The
phytohormone ABA is required for drought escape under long day (LD)
conditions by activating the transcription of florigen genes (Riboni et
al., 2013; Riboni, Robustelli Test, Galbiati, Tonelli, & Conti, 2016).
For example, the ABA-responsive element (ABRE)-binding factors ABF3 and
ABF4 induce SOC1 transcription and promote flowering under
drought conditions. Moreover, it has been shown that the activation of
the florigen genes HEADING DATE 3a and RICE FLOWERING LOCUS
T1 , and the floral integrator OsMADS50 (an ortholog of SOC1), modulate
the drought escape response through ABA-dependent and -independent
pathways in rice (Oryza sativa ) (Du et al., 2018). These studies
suggest that ABA is a positive regulator of flowering in the drought
escape response.
It has been shown that epigenetic mechanisms such as histone methylation
and acetylation play an important role in the plant response to
environmental conditions (Liu, Lu, Cui, & Cao, 2010; Luo et al., 2015).
Down-regulation of protein N-terminal acetylation enhances Arabidopsis
drought tolerance through the ABA pathway (Linster et al., 2015). The
equilibrium between histone acetyltransferases (HATs) and histone
deacetylases (HDACs) controls histone acetylation levels in nucleosomes,
and this modification can also affect chromatin structure and gene
expression in response to changes in the external environment
(Shahbazian & Grunstein, 2007; Yuan, Liu, Luo, Yang, & Wu, 2013).
Knocking out GCN5 , which encodes a member of the HAT family,
caused up-regulation of abiotic stress-inducible genes (Servet et al.,
2008); the histone acetyltransferases HAM1 and HAM2 increase HIS4
acetylation levels in FLC chromatin to promote FLCexpression and delay flowering time (Xiao et al., 2013). In Arabidopsis,
the elongator HAT complex is involved in ABA, drought, and oxidative
stress responses (Chen et al., 2006; Zhou, Hua, Chen, Zhou, & Gong,
2009; Versées, Groeve, & Lijsebettens, 2010). Histone deacetylase HDA5
promotes flowering time by repressing the expression of FLC andMAF1 (Luo et al., 2015). HDA9 delays flowering time by repressingAGL19 and thus promotes the expression of FT , especially
under SDs (Kang, Jin, Noh, & Noh, 2015; Kim et al., 2016). Knocking outHDA6 expression delayed flowering in both LD and SD conditions by
de-repressing FLC expression and reducing tolerance to ABA and
drought stress (Kim et al., 2017; Wu, Zhang, Zhou, Yu, & Chaikam, 2008;
Yu et al., 2011). Collectively, histone acetylation is involved in
flowering and abiotic stress signaling; however, how these regulatory
responses are coordinated via the various pathways, and the underlying
mechanisms, are largely unknown.
Controlling the timing of flowering is important during the response
stresses, but how the drought signal is integrated into the flowering
pathways is poorly understood at present. In this study, we report that
the expression of a Brassica rapa histone H4 gene (BrHIS4.A04,Bra036357 ) promotes flowering and decreases sensitivity to
drought under normal growth conditions. Under drought stress conditions,
the water deficit signal is probably translated to equilibrate the
acetylation level of BrHIS4 at certain ABA and photoperiodic flowering
gene loci, and therefore attenuates the expression to these genes to
prevent premature bolting in Chinese cabbage.
Materials and Methods
Plant materials and growth
conditions
A collection of 194 Chinese cabbage inbred lines used was used for
analysis of sequence variation and haplotyping of BrHIS4.A04.Detailed information on the 194 inbred lines is given in Su et al.
(2018.)
The Arabidopsis ecotype Col-0 (Columbia) was used as the wild-type in
this study. Arabidopsis seeds, including Col-0 and the BrHIS4.A04overexpression lines, were surface sterilized and incubated in the dark
at 4oC for 4 days. The seeds were then sown on 0.5X MS
salts medium solidified with 0.8% agar and grown at
22oC for 7 days. Seedlings were transferred to soil
and cultivated in a growth chamber on a 16 h light/8 h dark cycle at
22oC. For drought treatment, 3-week-old seedlings of
Arabidopsis or Chinese cabbage were deprived of water for 3 weeks. After
the drought treatment, the plants were re-watered.
Plasmid construction and plant
transformation
Total RNA was isolated from Chinese cabbage leaves using the RNAprep
Pure Plant Kit (DP441, Tiangen). First-strand cDNA was synthesized using
the Prime ScriptTM reagent kit with gDNA Eraser
(RR047A, Takara). The BrHIS4.A04 and BrVIN3.1 (Bra020445)
coding sequences were amplified by PCR using first-strand cDNA as
template. The coding sequences were then inserted into the p GADT7
and pGBKT7 plasmids, respectively. The BrHIS4.A04 coding sequence
was also inserted into the p MDC32 plasmid under control of the
CaMV35S promoter. Primers used for cDNA amplification are given in
Supplemental Table S1.
The p MDC32-BrHIS4.A04 recombinant plasmid was introduced intoAgrobacterium tumefaciens strain GV3101 and used for plant
transformation. The T1-generation transgenic plants were
selected on medium containing 15 mg/L hygromycin, and BrHIS4.A04expression was confirmed by semi-quantitative PCR. The primers used are
given in Supplemental Table S1.
Yeast two hybrid (Y2H)
assay
Young, healthy leaves from Chinese cabbage cultivar ‘BY’ (Yu, et al.,
2016) were collected and used for total RNA extraction. The Chinese
cabbage cDNA library in yeast was constructed using the Mate&Plate
Library Construction System (630490, Clontech). The BrVIN3.1coding sequence was inserted into p GBKT7 and transformed intoSaccharomyces cerevisiae strain Y2H. Transformants were selected
on SD medium with –Trp dropout supplement. Yeast-mating was utilized to
screen for BrVIN3-interacting proteins present in the Chinese cabbage
cDNA library.
p GADT7-BrHIS4.A04, p GBKT7-BrVIN3.1, and the negative
control plasmid were co-transformed into Saccharomyces cerevisiaeAH109 cells using the lithium acetate method according to the
manufacturer’s protocol (630489, Clontech). The co-transformants were
screened on double-dropout (-Trp/-Leu) medium at 30oC
for 2 days. To detect protein-protein interactions, positive
co-transformants were transferred to quadruple-dropout
(-Trp/-Leu/-His/-Ade) medium and incubated at 30oC for
4 days. Potential interactions were further confirmed by X-gal staining.
Gene expression analysis
For the analysis of BrHIS4.A04 expression during plant
development, young leaves of the Chinese cabbage inbred line ‘BY’ were
sampled at 10, 30, 50, 70, 90, and 120 days after planting in a growth
chamber (16 h light/8 h dark cycle at 22oC). For the
analysis of BrHIS4.A04 expression in different tissues,
seedlings, leaves, and roots were harvested from the 10-day plants,
while inflorescences, flowers, and stems were sampled from the early
flowering plants (~130 days after planting). For
analysis of the flowering genes FLC, GI, FT , and SOC1 ,
rosette leaves of 5-week-old Arabidopsis with or without drought
treatment were collected before flowering. For the analysis of drought-
and ABA-responsive genes, we collected rosette leaves of 6-week-old
Arabidopsis plants with or without drought treatment. Total RNA was
isolated and gene expression was quantified by Real-Time PCR (RT-PCR)
using SYBR Green PCR master mix (04887352001, Roche) on a LightCycler
480 RT-PCR system (Roche). Actin2 was used as the internal
control for normalization of gene expression. The primers used for
RT-PCR are given in Supplemental Table S1.
Western blot
Rosette leaves were collected from 5-week-old Arabidopsis plants and
ground to a powder in liquid nitrogen. The ground leaves were added to
extraction buffer (50 mM Tris-HCl, pH 7.5, 0.5 M sucrose, 1 mM
MgCl2, 1 mM EDTA, 5 mM DTT, 1 mM phenylmethanesulfonyl
fluoride and 1X protease inhibitor cocktail) and centrifuged at 14,000 g
for 30 min at 4oC. Soluble proteins in the supernatant
were fractionated by SDS-PAGE. Proteins were transferred to NC membrane
(A19465264, GE healthcare) using the Semi-Dry Electrophoretic Transfer
system (Bio-Rad). Anti-Histone H4 (ab10158, Abcam) and Anti-Histone H4
(acetyl K5+K8+K12+K16) (EPR16606, Abcam) antibodies were used as the
primary antibodies. IRDye 680RD Goat anti-rabbit antibodies (926-68071,
LI-COR) were used as the secondary antibody.
Chromatin Immunoprecipitation
ChIP experiments were performed using the Imprint Chromatin
Immunoprecipitation Kit (CHP1-96RXN, Sigma) according to the
manufacturer’s protocol. Rosette leaves were collected from 5-week-old
Arabidopsis plants and used for the ChIP experiments. Anti-Histone H4
(acetyl K5+K8+K12+K16) antibodies (EPR16606, Abcam) were used in the
immunoprecipitations and normal mouse IgG (M8695, Sigma Aldrich) was
used as the negative control. Quantitative measurements of gene
fragments were performed using SYBR Green PCR master mix (04887352001,
Roche) and the LightCycler 480 RT-PCR system (Roche). The Actin2gene was used as the internal gene expression control. The primers used
are given in Supplemental Table S1.
Water content and ion leakage rate
measurement
Rosette leaves from 6-week-old Arabidopsis seedlings were collected and
weighed (fresh weight; FW). The leaves were then incubated at
50oC for 7 days and weighed again (dry weight; DW).
Three replicates were measured for each transgenic line. Water content
was calculated as follows:
Water content (%) = (FW-DW)/FW
The seventh rosette leaves from 6-week-old Arabidopsis seedlings were
collected and incubated in 10 mL deionized water for 2 h. Conductance
was measured using a conductivity meter and denoted as S1. The leaf in
the 10 mL deionized water was then incubated at 100oC
for 30 min and cooled to room temperature. Conductance was again
measured and denoted as S0. Deionized water was used as the negative
control and the conductance measurements were denoted as SC and SC0. The
ion leakage rate was calculated as follows:
Ion leakage rate = (S1-SC)/(S0-SC0)
Measurements of stomatal density, stomatal aperture, and
guard cell
length
The middle section of a new, fully expanded rosette leaf was used to
count the stomata and for aperture and guard cell length measurements.
The abaxial epidermis was detached and photographed using a Nikon DS-RI
microscope. Stomatal aperture and guard cell length were measured using
Image J software, with at least 30 stomata measured from each transgenic
line. The guard cell number and pavement cell number on each photograph
were counted from at least 30 photographs of each transgenic line.
Stomatal density was calculated as follows:
Stomatal density=stomatal guard cell number/pavement cell number.
Determination of flowering time, silique length, and seed
number
Flowering time was determined by the number of rosette leaves present
when the first bud appeared. At least 15 individual plants of each
transgenic line were measured. Fully extended siliques from 7-week-old
Arabidopsis plants were photographed, and the lengths were measured
using Image J software. The number of seeds in each silique was then
counted. At least 20 siliques from each transgenic line were measured.
Results
BrHIS4.A04 interacts with
BrVN3.1
VIN3 binds to chromatin at the FLC locus (Sung & Amasino, 2004)
and interacts with members of the conserved polycomb-group repressive
complex 2 (PRC2) to perform its function (De Lucia, Crevillen, Jones,
Greb, & Dean, 2008; Wood et al., 2006). In Chinese cabbage, it has been
shown that one of the two VIN3 genes, BrVIN3.1 , was
selected for in the breeding history of a late-bolting ecotype, spring
Chinese cabbage (Su et al., 2018). To isolate proteins that interact
with BrVIN3.1, we constructed a B. rapa leaf cDNA library in
yeast. Using BrVIN3.1 as the ‘bait’ in a large-scale Y2H screening
experiment, we isolated 11 potential interacting partners (Table
1 and Figure 1 A, B ). Among these interactors, histone H4 has been
previously reported to function in flowering time regulation (He,
Michaels, & Amasino, 2003; Liu et al., 2010). Thus, we further verified
the interaction between BrHIS4.A04 and BrVIN3.1 in the one-to-one yeast
two-hybrid system (Figure 1 A, B ).
Overexpressing BrHIS4.A04 promotes
flowering in
Arabidopsis
HIS4 proteins are highly conserved in eukaryotes and show almost
identical amino acid sequences within and between species (Alvarez &
Loyola, 2017; Jiang & Berger, 2017; Sullivan & Landsman, 2003). In theBrassica rapa genome, there are 14 BrHIS4 homologs, of which
BrHIS4.A04 shares 87% amino acid sequence similarity with Arabidopsis
HIS4 (Supplementary Figure 1A, C ). To investigate the
expression patterns of these BrHIS4 genes, RNA-seq data was
generated from leaves harvested before flowering and during flowering
(Supplementary Figure 1B ). Eight of the 14 BrHIS4 genes
were detected (underlined in red in Supplementary Figure 1B ),
and we found that four of the eight (underlined in red and orange inSupplementary Figure 1B ) showed differential expression in the
two samples, including BrHIS4.A04 (Bra035673). RT-PCR analysis
was then performed on RNA extracted from multiple plant tissues at
different developmental stages to explore the expression pattern of
BrHIS4.A04 in the summer Chinese cabbage line ‘BY’ that can bolt within
~120 days without vernalization. Transcripts ofBrHIS4.A04 can be detected in all tissues or organs tested
(including root, stem, inflorescence, flower, and 10-day-old seedlings)
(Figure 1D ) and, moreover, we found that the expression ofBrHIS4.A04 changed dynamically during plant growth, with the
transcript level gradually rising prior to flowering and then decreasing
after flowering (Figure 1C ). To further study the function ofBrHIS4.A04 , the BrHIS4.A04 coding sequence was inserted
into the p MDC32 plasmid under control of the CaMV35S promoter and
transformed into Arabidopsis Col-0. Two individual transgenic lines
(hereinafter designated BrHIS4.A04OE) were used in a
study in which the relative expression of BrHIS4.A04 was
significantly increased (Supplementary Figure 2 ). HomozygousBrHIS4.A04OE plants showed no obvious
morphological differences compared to wild-type plants at various stages
of development (Figure 1E and Supplementary Figure 3 ). Both
transgenic lines displayed normal rosette leaf development, and seed set
was also found to be normal (Figure 1E and Supplementary Figure
3 ). However, in three independent experiments, we observed thatBrHIS4.A04OE plants display early flowering
under normal LD conditions (Figure 1E, F ). More specifically,BrHIS4.A04OE plants flowered earlier (at
~6 rosette leaves) than did the control, and this
difference was statistically significant (Figure 1F ).
FLC acts as a floral repressor and inhibits flowering by directly
repressing the promoters of the key flowering genes FT andSOC1 (Lee et al., 2000). qRT-PCR analysis showed a significant
decrease in expression of FLC , whereas the expression of the
flowering activator FT increased inBrHIS4.A04OE plants (Figure 1G, H ).
These results suggest that BrHIS4.A04 negatively regulates FLCexpression, and that it is also a positive regulator of FT .
Overexpression of BrHIS4.A04confers drought hypersensitivity
Flowering is initiated in response to the integration of both
environmental cues and endogenous pathways. We noticed that in
Arabidopsis, AtHIS4 can be slightly induced by a 24-hour drought
treatment in roots, but not by any other abiotic stresses, such as
osmotic, salt, and heat stress
(https://www.arabidopsis.org/servlets/TairObject?id=33482&type=locus).
We thus exposed plants of the Chinese cabbage line BY to different
abiotic stresses to investigate the expression profile ofBrHIS4.A04 under osmotic, salt, heat, and drought stress
conditions. We found that BrHIS4.A04 expression is induced by
drought, suppressed by heat, and is unaffected under osmotic and salt
stress conditions (Supplementary Figure 4 ). Moreover, the
phenotypes of BrHIS4.A04OE and Col-0 plants in
response to these stresses were recorded, and we found that theBrHIS4.A04OE plants are hypersensitive to
drought in comparison with Col-0 (Figure 2A ). There were no
differences in the water content and ion leakage rate between theBrHIS4.A04OE and Col-0 plants under normal
growth conditions; however, after drought stress, the water content ofBrHIS4.A04OE plants was reduced to
~70% while the ion leakage rate increased to
~200% of that of the control plants (Figure 2B,
C ).
We then examined expression of the drought response marker geneRD29b (Yang et al., 2011). Under normal conditions, no difference
in expression was found between the Col-0 andBrH4IS.A04OE plants. After drought treatment,RD29b expression was induced in both plants; however, the
relative induction level was much lower inBrHIS4.A04OE plants (Figure 2D ). It
was previously reported that drought stress is accompanied by reactive
oxygen species (ROS)generation (Gechev, Dinakar, Benina, Toneva, &
Bartels, 2012). Expression of the SOD , POD , andCAT12 genes that encode ROS scavenging enzymes was also detected
under the different conditions. As we observed at RD29b , the
relative degree of induction for all of the above three genes was less
in the BrHIS4.A04OE plants compared with the
control plants (Figure 2E-I ). Taken together, these results
suggest that the expression of BrHIS4.A04 can attenuate the
drought response in plants exposed to water deficit conditions.
BrHIS4.A04 leads to drought hypersensitivity
independently of
ABA
The plant response to drought is highly correlated with stomata because
leaf water loss through transpiration is controlled by stomatal
development (including the size and density of stomata on the epidermis)
and behavior (the stomatal aperture) (Buckley, Sack, & Farquhar, 2017).
We therefore first investigated stomatal size and density to understand
the reason for the higher water loss observed inBrHIS4.A04OE plants; however, no difference was
found between the BrHIS4.A04OE and Col-0 plants
with or without drought treatment (Figure 3A-H ). Stomatal
movement was then measured, and the results showed that drought induced
dramatic stomatal closure in wild-type plants, but the stomatal
apertures were only slightly decreased inBrHIS4.A04OE plants (Figure 3I ).
ABA is an important mediator between drought stress and stomatal
movement (Leung & Giraudat, 1998; Lim, Baek, Jung, Kim, & Lee, 2015).
To determine whether BrHIS4.A04 induces drought hypersensitivity through
the ABA pathway, genes involved in ABA biosynthesis (ABA1 andNCED3 ) and signaling (ABI1 and MYB2 ) were analyzed.
As in the drought response genes described above, the expression of the
four ABA genes was higher in BrHIS4.A04OEplants compared with Col-0 under normal conditions, and expression was
induced in both plants after drought treatment. However, the induction
was to a much lesser degree in BrHIS4.A04OEplants (Figure 3J-M ). These results suggest that overexpressingBrHIS4.A04 in Chinese cabbage leads to drought hypersensitivity
dependently of ABA.
BrHIS4.A04 prevents premature flowering under drought
conditions
Since BrHIS4.A04 is involved in both the drought response and flowering
control, we asked whether BrHIS4.A04 plays a role in integrating the
drought response with flowering regulation. The flowering times of theBrHIS4.A04OE and Col-0 plants grown under
drought stress were then recorded (Figure 4A, B ). We found that
the drought-treated Col-0 plants bolted earlier than plants grown under
normal conditions as expected; however, the drought-treatedBrHIS4.A04OE plants bolted at roughly the same
time, if not slightly later than, the plants grown under normal
condition (Figure 4B ). This result showed that drought had a
much stronger effect on wild-type plants, but no effect or only a slight
effect on the BrHIS4.A04OE plants.
We next examined the expression of FLC , GI , FT , andSOC1 in plants grown under both normal and drought conditions.
Under normal conditions, the over-expression of BrHIS4.A04resulted in increased levels of GI , FT and SOC1transcription (Figure 4D-E ), and slightly reduced levels ofFLC -specific mRNA (Figure 4F ), suggesting that the early
flowering of BrHIS4.A04OE plants grown under
normal conditions mainly depends on the photoperiodic flowering pathway,
which was also reported by Riboni et al. (2016). After drought
treatment, a slight reduction in FLC expression and increases in
the expression of GI , FT and SOC1 were found in
Col-0 plants (Figure 4C-F ) as reported by Riboni et al.
(2013). Intriguingly, however, we found that there was no
increase in the expression of the GI , FT , and SOC1genes in BrHIS4.A04OE plants (Figure
4D-F ), which is consistent with the observation that there was no
difference in flowering time between the normally-grown and
drought-treated BrHIS4.A04OE plants. These
results suggest that BrHIS4.A04 can prevent premature flowering in
Chinese cabbage under drought conditions, mainly by maintaining the
expression of photoperiodic flowering genes, which is further supported
by the fact that although expression of FLC was dramatically
induced after drought in BrHIS4.A04OE plants,
the flowering time did not change (Figure 4B, C ).
Overexpressing BrHIS4.A04 leads to
a higher chromatin acetylation level in plants
It has been widely reported that histone H4 acetylation is involved in
regulating flowering time and the drought response (He et al., 2003).
Considering the possibility that BrHIS4.A04 may be one of the hubs of
the two physiological activities, we wondered whether the two processes
are coordinated via the acetylation of histone H4. Total protein was
then extracted from the leaves of plants grown with or without drought
treatment, and a crude profile of H4 acetylation level was determined by
Western blotting using an anti-H4 (acetyl K5+K8+K12+K16) antibody. Under
normal growth conditions, a higher level of H4 acetylation was found inBrHIS4.A04OE plants in keeping with its higher
histone H4 protein level detected using the anti-H4 antibody. After
drought treatment, the H4 acetylation level increased dramatically in
Col-0 plants as expected; however, no increase was found inBrHIS4.A04OE plants (Figure 5A ).
We then examined H4 acetylation at four genetic loci; the ABA genesABI1 and MYC2 and the flowering genes FLC andFT . Chromatin immunoprecipitation assays with anti-H4 (acetyl
K5+K8+K12+K16) antibody were performed followed by RT-PCR
(Figure 5B-E and Supplementary Figure 5 ). Under normal growth
conditions, the levels of H4 acetylation at all the tested loci were
much higher in BrHIS4.A04OE plants when
compared with Col-0 plants (Figure 5B-E ). This result is
consistent with their expression profiles and the consequent
early-flowering and drought-sensitive phenotypes of theBrHIS4.A04OE plants. After drought treatment,
the H4 acetylation levels of these genes increased dramatically in Col-0
plants, but remained at about the same level inBrHIS4.A04OE plants grown under both normal and
drought conditions (Figure 5B-E ). This observation is in
keeping with the hypothesis that BrHIS4.A04 can prevent premature
flowering by attenuating the expression of drought and photoperiodic
flowering genes.
Discussion
BrHIS4.A04 is a non-canonical histone
H4
In the nuclei of eukaryotic cells, chromatin is the physiological
template for various genetic processes. The basic unit of chromatin is
the nucleosome, a structure composed of an octamer of the core histone
proteins H2A, H2B, H3, and H4, around which is wrapped 146 base pairs of
chromosomal DNA. These proteins are highly conserved across eukaryotic
species (Alvarez & Loyola, 2017; Sullivan & Landsman, 2003; Weber &
Henikoff, 2014). All four types of histones are encoded by multiple
genes; for example, there are eight AtHIS4 genes with different
nucleotide sequences, but all eight genes encode single proteins with
identical amino acid sequences, suggesting its conserved role in
evolution (Tenea et al., 2009). Protein sequence alignment revealed that
the protein encoded by BrHIS4.A04 shares 87% amino acid sequence
homology with that encoded by AtHIS4 (Supplementary
Figure 1C ). Furthermore, we found that the expression ofBrHIS4.A04 is induced during growth and by drought in Chinese
cabbage (Figure 1C, Supplementary Figure 3 ). Thus, the
conserved features of BrHIS4.A04 and its induced expression in
response to drought, together with the functional studies shown in the
Results section hint at its non-canonical role compared to other BrHIS4
proteins. Histones function to control the structure and accessibility
of the chromatin environment by altering the biochemical properties of
the nucleosome or through the recruitment of distinct binding partners
(Jiang & Berger, 2017; Melters et al., 2019; Weber & Henikoff, 2014).
One such method of histone-mediated control comes from the exchange of
canonical histones with non-allelic histone variants, which alter the
fundamental structure and stability of the nucleosome. H2A.Z is one of
the most enigmatic of these histone variants (Kawashima et al., 2015).
It has been reported that H2A.Z within gene bodies is correlated with
genes that respond to environmental stresses (Dong et al., 2018; Sura et
al., 2017). One potential scenario is that BrHIS4.A04 gained some
similar features of H2A.Z in the evolution of Chinese cabbage, and the
amino acid variant of BrHIS4.A04 might facilitate it to specially target
some environmentally responsive genes. However this hypothesis needs to
be further tested by comparing the genome-wide target genes of
BrHIS4.A04 and other BrHIS4 homologues which can be identified by the
ChIP-seq method.
H4 acetylation is one of the hubs that connects the
drought response and flowering
regulation
Resistance to drought involves global reprogramming of transcription,
hormone signaling, and chromatin modification in plants (Fang & Xiong,
2014). However, the ways in which these regulatory responses are
coordinated via the various pathways are largely unknown. Hwang et al.
(2019) reported that the ABA-responsive element (ABRE)-binding factors
ABF3 and ABF4 are rapidly induced when the plant is exposed to drought
stress, and that ABF3 and ABF4 interact with NF-YCs to promote flowering
by inducing SOC1 transcription. Here, together with our findings, we
report an essential drought-responsive network in which plants trigger
the ABA signaling to stimulate flowering to confer drought response, and
the change in H4 acetylation at certain ABA and flowering gene loci is
the hub of the network. In Arabidopsis, we observed that mutation of
histone H4 deacetylase HDA6 resulted in enhanced drought tolerance and
late flowering (Wu et al., 2008). Further study showed that HDA6
directly represses the acetate biosynthesis pathway under normal
conditions (Kim et al., 2017). In addition, exogenous acetic acid can be
converted to acetyl-CoA and used as a substrate for histone acetylation
to increase the acetyl-H4 level and boost drought tolerance in plants.
Therefore, we proposed that the drought-induced activation of acetate
biosynthesis potentially leads to genome-wide H4 acetylation, which
connects fundamental metabolism, epigenetic regulation, and hormone
signaling, and ultimately affects plant environmental adaptation.
Besides, compared with chromatin modifiers, there are quite a few
studies that discuss the role of their histone targets, except for
H2A.Z, in which they are used as epigenetic indicators in most cases. H4
acetylation is often associated with chromatin remodeling during gene
activation, but as far as we know, there are no studies that describe
the effect of H4 acetylation on FT expression. The results of our
study suggest that H4 acetylation positively regulates the expression ofFT , and a proper acetylation of BrHIS4 at the FT locus can
keep it functioning within a reasonable range.
BrHIS4.A04 is selected in Chinese cabbage
breeding?
Mining elite alleles for drought resistance and late flowering is
important for the improvement of cultivated Chinese cabbage and
selection for market demand. Sequence analysis of BrHIS4.A04showed 28 SNPs in the gene body, with 24 SNPs in the promoter and the
other four in the coding region that did not cause changes in the amino
acid sequence. To gain further insight into the allelic differentiation
of BrHIS4.A04 , we extended our study in the 194 Chinese cabbage
accessions reported by Su et al. (2018, 2019). Two haplotypes ofBrHIS4.A04 were identified in the 194 lines; Hap1 was the major
haplotype present in 182 accessions, while only 12 accessions carry Hap2
(Supplementary Figure 6 A, B). Phylogenetic analysis showed that
the 194 line can be classified into four different groups (Su et al.,
2018)
The Hap1-carrying lines were found to be distributed among all four
groups, while the Hap2 lines were only present in the relatively
primitive Aut1 and summer groups (Supplementary Figure 6C ).
Taking geography into account, we noticed that seven of the 12 Hap2
lines originate from Tianjin, China (Supplementary Figure 6C,
red circles ). These results are consistent with the fact that Chinese
cabbage germplasm from Tianjin has been reported to be salt and drought
tolerant due to its long breeding history on mildly saline and alkaline
soils. The drought tolerance of the seven lines was further studied, and
they indeed showed higher survival rates after rehydration
(Supplementary Figure 6 D). However, the significance of this
result can be statistically evaluated only after more varieties from
Tianjin are collected and introduced into the study.
Taken together, we report here that BrHIS4.A04 can prevent premature
flowering mainly through its action on the photoperiodic flowering
pathway by attenuating ABA signaling under drought conditions. SinceBrHIS4.A04OE plants displayed no phenotypes
related to vegetative and reproductive development in response to
drought, we think that our findings will contribute to the fine-tuning
of flowering time in crops with no growth penalty by genetic engineering
or other breeding methods.