A rapid and robust leaf ablation method to visualize bundle sheath cells and chloroplasts in C3 and C4 grasses

Abstract

Background:
It has been proposed that engineering the C4 photosynthetic pathway into C3 crops could significantly increase yield. This goal requires an increase in the chloroplast compartment of bundle sheath cells in C3 species. To facilitate large-scale testing of candidate regulators of chloroplast development in the rice bundle sheath, a simple and robust method to phenotype this tissue in C3 species is required.

Results:
We established a leaf ablation method to accelerate phenotyping of rice bundle sheath cells. The bundle sheath cells and chloroplasts were visualized using light and confocal laser microscopy. Bundle sheath cell dimensions, chloroplast area and chloroplast number per cell were measured from the images obtained by confocal laser microscopy. Bundle sheath cell dimensions of maize were also measured and compared with rice. Our data show that bundle sheath width but not length significantly differed between C3 rice and C4 maize. Comparison of paradermal versus transverse bundle sheath cell width indicated that bundle sheath cells were intact after leaf ablation. Moreover, comparisons of planar chloroplast areas and chloroplast numbers per bundle sheath cell between wild-type and transgenic rice lines expressing the maize GOLDEN-2 (ZmG2) showed that the leaf ablation method allowed differences in chloroplast parameters to be detected.

Conclusions:
Leaf ablation is a simple approach to accessing bundle sheath cell files in C3 species. We show that this method is suitable for obtaining parameters associated with bundle sheath cell size, chloroplast area and chloroplast number per cell.

Full text

METHODOLOGY Open Access
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Billakurthi and Hibberd Plant Methods (2023) 19:69
https://doi.org/10.1186/s13007-023-01041-x Plant Methods
*Correspondence:
Kumari Billakurthi
kb720@cam.ac.uk
1Department of Plant Sciences, University of Cambridge, Downing Street,
Cambridge, UK
Abstract
Background It has been proposed that engineering the C4 photosynthetic pathway into C3 crops could significantly
increase yield. This goal requires an increase in the chloroplast compartment of bundle sheath cells in C3 species. To
facilitate large-scale testing of candidate regulators of chloroplast development in the rice bundle sheath, a simple
and robust method to phenotype this tissue in C3 species is required.
Results We established a leaf ablation method to accelerate phenotyping of rice bundle sheath cells. The bundle
sheath cells and chloroplasts were visualized using light and confocal laser microscopy. Bundle sheath cell
dimensions, chloroplast area and chloroplast number per cell were measured from the images obtained by confocal
laser microscopy. Bundle sheath cell dimensions of maize were also measured and compared with rice. Our data
show that bundle sheath width but not length significantly differed between C3 rice and C4 maize. Comparison
of paradermal versus transverse bundle sheath cell width indicated that bundle sheath cells were intact after leaf
ablation. Moreover, comparisons of planar chloroplast areas and chloroplast numbers per bundle sheath cell between
wild-type and transgenic rice lines expressing the maize GOLDEN-2 (ZmG2) showed that the leaf ablation method
allowed differences in chloroplast parameters to be detected.
Conclusions Leaf ablation is a simple approach to accessing bundle sheath cell files in C3 species. We show that this
method is suitable for obtaining parameters associated with bundle sheath cell size, chloroplast area and chloroplast
number per cell.
Keywords Oryza sativa, Leaf ablation, Bundle sheath cells, Chloroplasts, Confocal microscopy
A rapid and robust leaf ablation
method to visualize bundle sheath cells
and chloroplasts in C3 and C4 grasses
Kumari Billakurthi1* and Julian M. Hibberd1
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Billakurthi and Hibberd Plant Methods (2023) 19:69
Background
Photosynthesis is fundamental to life on earth and allows
assimilation of atmospheric CO2 into biomass via the
Calvin-Benson-Bassham or C3 cycle [1–3]. In plants
the photosynthetic process is broadly categorised into
C3, C4 and Crassulacean Acid Metabolism based on the
pathway of carbon xation. However, plants that use
C3 photosynthesis predominate such that species using
C4 and Crassulacean Acid Metabolism account for only
three and six% of land plants respectively [4–6]. In C3
plants, mesophyll cells are lled with chloroplasts and
so are the major site of photosynthesis (Fig.1a). In these
plants the enzyme Ribulose-1,5-Bisphosphate Carboxyl-
ase/Oxygenase (RuBisCO) carboxylates the ve-carbon
compound Ribulose-1,5-bisphosphate (RubP) via the
C3 cycle to generate two molecules of the three-carbon
compound 3-phosphoglycerate. In contrast, in the vast
majority of C4 plants the reactions of carbon assimilation
are equally partitioned between mesophyll and bundle
sheath cells. HCO3 − 1 is initially xed in mesophyll cells
by Phosphoenolpyruvate Carboxylase (PEPC) to generate
four-carbon compounds such as malate and aspartate
that then diuse into bundle sheath cells. Decarboxyl-
ation of either aspartate or malate in the bundle sheath
releases high concentrations of CO2 in bundle sheath
cells that can then be assimilated by RuBisCO [7].
Due to the C4 cycle concentrating CO2 around
RuBisCO, C4 species are more ecient under dry and
high-temperature conditions. Moreover, they often have
improved water and nitrogen use eciencies compared
with C3 plants [8–11]. Apart from those species that use
single-celled C4 photosynthesis [12], a unifying character
underpinning the C4 pathway is a specialised form of leaf
morphology termed Kranz anatomy [13]. Kranz anatomy
is characterised by a high vein density and bundle sheath
cells that are altered both morphologically but also in
terms of organelle occupancy and positioning. During
the C3 to C4 trajectory, in some lineages while not always
the case, evolution has generated bundle sheath cells that
are larger in the medio-lateral leaf axis [14, 15] and con-
tain numerous larger chloroplasts ([16], Fig.1b).
Increasing the photosynthetic eciency of C3 crops
would help meet future demands for food, especially
under changing climatic conditions. It has been pre-
dicted that introducing the C4 pathway into C3 crops
could increase their photosynthetic eciency by up to
50% [17]. However, one of the main bottlenecks is an
incomplete understanding of how bundle sheath cells
become photosynthetically activated in C4 plants. On
average, the bundle sheath chloroplast content of C4 spe-
cies is ~ 30% more than in C3 species [16, 18], but how
this evolved is not fully understood. e GOLDEN2-
LIKE family of transcription factors known to regulate
chloroplast development in C4 species [19–21]. Although
overexpression of GOLDEN2 or GOLDEN2-LIKE 1 from
C4Zea mays in rice increased bundle sheath chloroplast
volume, this did not phenocopy the increase in chloro-
plast occupancy found in C4 plants [22].
Introducing C4 bundle sheath anatomy into C3 rice is
therefore likely to involve large-scale testing of candi-
date genes involved in bundle sheath cell and chloroplast
development and phenotyping bundle sheath cells. How-
ever, the bundle sheath has been challenging to pheno-
type in C3 plants. Classical bright-eld light microscopy
after embedding samples in resin and thin sectioning
has been used [18]. Although this is simple and easily
available, it only captures two-dimensional (2D) infor-
mation from a thin section. 2D-transmission electron
microscopy (2D-TEM) is widely used for characterising
the ultracellular structure and organisation in photo-
synthetic cell types [23] but it is expensive and has the
same limitations as light microscopy when cell and chlo-
roplast parameters are being quantied. A single-cell
isolation method has been established to study meso-
phyll and bundle-sheath cell dimensions and chloroplast
Fig. 1 Confocal laser scanning microscopy images of C3 (rice) and C4
(maize) mesophyll and bundle sheath cells. Images are derived from para-
dermal sections. Representative maximum intensity projection image of a
Z-stack from wild type rice (a) and maize (b). Bundle sheath and mesophyll
cells are highlighted with white and blue lines respectively. Chloroplasts
from bundle sheath cells of maize generate lower autofluorescence due
to lower amounts of Photosystem II. Cp: Chloroplasts (pseudo colour:
magenta)
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Billakurthi and Hibberd Plant Methods (2023) 19:69
occupancy, but it requires enzymatic digestion of leaf
tissue that might disturb cell integrity and chloroplast
size [22]. Mechanical isolation of bundle sheath strands
has previously been used for C4 grasses [24, 25]. But, a
number of chemical treatments are involved, and suc-
cess depends on the correct preparation of leaf tissue as
well as optimisation of the grinding procedure. More-
over, vein positional information is typically lost mean-
ing that it is challenging to dene the origin of bundle
sheath cells. Furthermore, application of this method to
C3 grasses might not be feasible due to many mesophyll
layers. Lastly, more advanced electron microscopy-based
3D reconstruction methods such as serial block-face
scanning electron microscopy (SBF-SEM) can cover large
elds of view and reconstruct ultrastructural features in
3D such that volume of leaf cells and chloroplasts can be
quantied [26]. However, it is costly and labour-intensive.
us, each of these approaches has disadvantages for
high-throughput screening of bundle sheath cells in spe-
cies such as C3 rice.
To address this, we established a simple and robust
method to expose bundle sheath cell les in rice and
measure their cell dimensions, as well as the planar chlo-
roplast area and chloroplast number per cell. We show
that these bundle sheath cells are intact and the chlo-
roplast number per cell is comparable with previous
reports [22]. We also applied this method to the C4 spe-
cies maize to measure bundle sheath cell dimensions and
made comparisons between bundle sheath cells in these
two species. When combined with genetic perturbations
we anticipate that this approach will provide insight into
structure function relations of bundle sheath cells in spe-
cies such as rice.
Results
A simple and robust method to visualize bundle sheath
cells in C3 rice and C4 maize
e middle region of fully expanded fourth leaves from
rice and maize was xed with glutaraldehyde. Prior to
ablation, although parallel venation was detectable in rice
at low magnication, when higher power objectives were
used the signicant amount of light scattering meant that
individual cells including the bundle sheath were not vis-
ible (Fig.2a, c). However, bundle sheath strands and cells
became visible (Fig.2b, d) after the adaxial side of leaves
was ablated by gentle scraping (Additional le 1). In rice
scraping was carried out until mesophyll tissue surround-
ing intermediate veins appeared less green. As the bundle
sheath is deep in the C3 leaf because of the many layers
of mesophyll cells [27], two to three minutes of ablation
(Additional le 1) was required to expose bundle sheath
cells around intermediate veins (Fig.2b, d). Consistent
with rice leaf anatomy, three to four intermediate veins
(rank-1; tertiary; 3°) were present between the larger lat-
eral (secondary; 2°) veins.
In maize, dark green strands that represent the bun-
dle sheath were visible prior to scraping (Fig. 2e) and
although mesophyll cells were detectable at higher
magnication this was not true for the bundle sheath
(Fig.2g). Scraping of maize allowed les of dark green
bundle sheath and the less green mesophyll cells to be
identied (Fig.2f). C4 maize has increased numbers of
intermediate (rank-1 + rank-2) veins between the larger
laterals because of an increase in the density of rank-2
intermediates [28] and leaf ablation was consistent with
this (Fig.2f). In C4 maize it took less than one minute to
ablate mesophyll layers such that bundle sheath cell les
were clearly visible (Fig.2f, h).
Quantication of bundle sheath cell dimensions
To provide quantitative insight into dierences between
bundle sheath cells of C3 rice and C4 maize we ablated
leaf tissue from each species and then used calcouor
white to mark cell walls (Fig.3a, b). Bundle sheath cell
length and width measurements were taken at the mid-
point of both the proximal-distal and medio-lateral axes,
and planar cell area was calculated (Fig.3a, b). Average
bundle sheath cell width was 15μm in rice and 32μm in
maize (Fig.3c) but there is a very small dierence in bun-
dle sheath cell length between these two species (Fig.3d).
However, as a consequence of the increased width of
bundle sheath cells in maize, mean bundle sheath cell
area was signicantly higher (1404 µm2) than that of
rice (598 µm2) (Fig.3e). We also observed high variance
in bundle sheath cell dimensions in both rice and maize.
is variation in width and length of bundle sheath cells
was two-fold and three-fold respectively in both species
(Fig.3c, d), and the variation in bundle sheath cell area
of maize was around 1.4 times greater than that of rice
(Fig.3e).
Visualisation and quantication of chloroplast parameters
in rice bundle sheath cells
We next wished to investigate whether bundle sheath
chloroplast number and size could be determined after
leaf ablation. Transgenic rice lines expressing the maize
GOLDEN2 (ZmG2) transcription factor under the con-
trol of the maize ubiquitin promoter are known to con-
tain larger chloroplasts [22] and so were used as controls.
We used calcouor white to stain cell walls and chloro-
phyll autouorescence to visualize bundle sheath cell
chloroplasts. Z-stacks of 82 and 90 bundle sheath cells
from wild-type and pZmUbi::ZmG2 rice respectively
were acquired by confocal laser scanning microscopy.
Maximum intensity projection images (Fig.4a) were used
to quantify individual chloroplast areas and chloroplast
number per cells. e average planar area of individual
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Billakurthi and Hibberd Plant Methods (2023) 19:69
bundle sheath chloroplasts in wild-type was ~ 17 µm2
(with a range from 7 to 37 µm2; Fig.4b). Consistent with
published data [22] planar area of bundle sheath chloro-
plasts was signicantly increased in the pZmUbi::ZmG2
line and ranged from 7 to 54 µm2 (Fig.4b). Moreover,
as expected [22] there was no dierence in chloroplast
numbers in the bundle sheath between controls and
pZmUbi::ZmG2 (Fig. 4c). However, total chloroplast
occupancy of bundle sheath cells in pZmUbi::ZmG2 was
signicantly increased due to the greater planar area of
Fig. 2 Visualization of rice and maize leaves before and after ablation. Light microscopy images of rice (a–d) and maize (e–h) leaves before (left) and after
scraping (right). Low power images (b, f) illustrating the impact of ablation on bundle sheath visibility (highlighted with white lines). Representative im-
ages of selected regions at higher magnification (d, h). Bundle sheath cells highlighted with white lines. Abbreviations are as follows: 2°: secondary veins;
3°: tertiary veins; V: Vein; BS: Bundle sheath cell. M: Mesophyll tissue. Scale bar represents 100 μm (a, b, e, f) and 20 μm (c, d, g, h)
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Billakurthi and Hibberd Plant Methods (2023) 19:69
individual chloroplasts (Fig. 4d). Further, total chloro-
plast number per bundle sheath cell (with a range from 8
to 25) obtained from leaf ablation (Fig.4c) was compara-
ble with that previously reported from analysis of isolated
single-cells (with a range from 6 to 21; [22]). erefore,
gentle and careful ablation can be used to obtain accurate
estimates of chloroplast numbers in rice bundle sheath
cells.
Acquisition of three-dimensional (3D) images is of
course more time consuming than two-dimensional (2D)
images. We therefore wanted to test if there was a dier-
ence between bundle sheath chloroplast numbers esti-
mated by the two approaches and so obtained 2D and
3D images of the same 31 cells from wild-type (Fig.5a).
ese data showed that the bundle sheath chloroplast
number was signicantly higher (Fig.5b) when estimated
from 3D imaging (with a range from 11 to 25) compared
with 2D imaging (with a range from 10 to 19). However,
planar area of individual chloroplasts in bundle sheath
cells was not dierent between the two datasets (Fig.5c).
We conclude that 3D imaging provides a more precise
estimate of bundle sheath chloroplast numbers but either
method can be used to quantify chloroplast size.
The relationship between bundle sheath paradermal cell
area and chloroplasts
We wanted to use the above data to understand the rela-
tionship between bundle sheath chloroplast occupancy
and cell area in rice. erefore, a simple linear regression
model was performed between bundle sheath paradermal
Fig. 3 Quantification of bundle sheath cell dimensions in rice and maize. a Representative confocal laser scanning microscopy images of calcofluor white
stained (pseudo colour: grey) bundle sheath strand and mesophyll tissue of rice (a) or maize (b). Images were cropped to focus on a bundle sheath strand.
Violin plots representing bundle sheath cell cell width (c), length (d) and area (e) in rice and maize. Blue dot represents mean values. Bundle sheath cell
length and width measurements were taken at the mid-point of the proximal-distal and medio-lateral axes respectively (annotated with white arrows in
a and b). Each observation represents one cell. Number of cells (n) = 82 and 90 from rice and maize, respectively. Abbreviations are as follows: V: Vein; BS:
Bundle sheath cell; M: Mesophyll cell. Statistical test: t-test
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Billakurthi and Hibberd Plant Methods (2023) 19:69
cell area and chloroplast size and number. is showed
that the average planar and maximum chloroplast area
per cell did not vary with bundle sheath cell area (Fig.6a,
b). But, chloroplast number and thus total chloroplast
area per cell increased with cell area (Fig.6c and d). e
percentage of cell area occupied by chloroplasts was neg-
atively correlated with bundle sheath cell area (Fig.6e).
Discussion
It is widely recognised that improving photosynthesis
in crops is one mechanism to improve yield [29]. One
approach that has been proposed [17, 30] is to engi-
neer the C4 pathway into C3 crops such as rice and it is
estimated that this could improve yields by up to 50%.
However, this goal is challenging and would require a
signicant increase in the chloroplast compartment of
bundle sheath cells from C3 crops such as rice. It has
Fig. 5 Comparison of chloroplast numbers obtained from two and three-dimensional (2D and 3D) imaging. a Representative two-dimensional (left) and
three-dimensional (right) images of wild-type rice leaves. Bundle sheath cells are highlighted with white lines. Chloroplasts present in the 3D image but
not detected in the 2D image are highlighted with white arrows. b Chloroplast number per bundle sheath cell - each observation represents one cell
(n = 31). c Planar chloroplast area of bundle sheath cells - each observation represents one chloroplast. Number of chloroplasts (n) = 236 and 242 from 2D
and 3D imaging, respectively. Blue dot in the violin plots represent mean values. Statistical test: t-test. M: Mesophyll tissue
Fig. 4 Visualization and quantification of chloroplast area and number in the bundle sheath of rice. a Representative maximum intensity projection
image of a Z-stack from wild-type and the GOLDEN2 overexpressing line (pZmUbi::ZmG2). Images were cropped to focus on a bundle sheath strand.
Bundle sheath cells are highlighted with white lines. b Planar area of individual chloroplasts from bundle sheath cells. Each observation represents one
chloroplast. Number of chloroplasts (n) = 1032 and 1114 from wild-type and pZmUbi::ZmG2 rice lines, respectively. c Chloroplast number per bundle
sheath cell. Each observation represents one cell. d Total chloroplast area per bundle sheath cell. Each observation represents the total chloroplast area
of a cell. Number of cells (n) = 82 and 90 from wild-type and pZmUbi::ZmG2 rice lines, respectively. Blue dot in the violin plots represent mean values.
Statistical test: t-test
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Billakurthi and Hibberd Plant Methods (2023) 19:69
been challenging to phenotype bundle sheath tissue in C3
species as these cells are deeper in the leaf because of the
many layers of mesophyll cells [27]. Approaches includ-
ing bright-eld light microscopy [18], transmission elec-
tron microscopy [23], serial block-face scanning electron
microscopy [26] and single-cell isolation methods [22]
are slow and so this hinders rapid analysis of transgenic
lines harbouring candidate genes that are hypothesized
to control chloroplast proliferation in the bundle sheath.
To this end, we sought to establish a rapid and robust
method to visualize bundle sheath cell les in C3 rice.
Including sample preparation time, the ablation
method reported here requires about 30min to pheno-
type one leaf sample and can capture images from 30 to
40 bundle sheath cells in one focal plane. To obtain three-
dimensional imaging via acquisition of z-stacks approxi-
mately one hour is needed. is compares favourably
with other approaches such as the published single-cell
isolation method [22] which involves ve hours of sam-
ple preparation followed by three-four hours to image a
similar number of cells. us, we estimate that the leaf
ablation method is at least ten times faster than single-
cell isolation. Other methods that involve resin-embed-
ding, thin-sectioning, and then image capture via light or
electron microscopy take a few weeks. e leaf ablation
method also excludes hazardous chemicals and enzymes
for sample preparation, and it is noteworthy that it also
allows specic vein types to be identied prior to imag-
ing, which can be challenging with the single-cell isola-
tion method as the leaf tissue is subject to enzymatic
digestion. We therefore consider this simple ablation
approach to be robust and useful for high-throughput in
vivo phenotyping of bundle sheath cells in C3 species.
To provide evidence that imaging after ablation cap-
tures parameters derived from intact bundle sheath cells,
the width of rice bundle sheath cells was measured from
transverse sections obtained from serial block-face scan-
ning electron microscopy (Additional le 2a) and com-
pared with paradermal cell width obtained from confocal
microscopy imaging after leaf ablation (Additional le
2b). As bundle sheath cells are cylindrical in rice [14],
the width should equal the depth. In fact, mean bundle
sheath cell width was lower (~ 10 μm) when estimated
from transverse sections compared with paradermal sec-
tions (~ 15μm; Additional le 2b) implying that the esti-
mates of cell width after ablation are not associated with
incomplete imaging of this cell type. It is also possible
that paradermal sections preferentially captured informa-
tion on bundle sheath cells lateral to each vein (Fig.3a).
It has been reported that during the C3 to C4 trajectory
bundle sheath cells elongate less along the axis of the
vein but become wider [14, 15]. Consistent with this,
we observed a two-fold increase in bundle sheath cell
width in maize compared with rice (Fig. 3c). However,
the length of bundle sheath cells in the two species were
similar and so these data suggest that a reduction in bun-
dle sheath cell length may not be required for the evo-
lution of C4 photosynthesis. e average bundle sheath
cell length in maize is similar to what has been previously
reported [14]. However, they reported higher values for
average rice bundle sheath cell length (~ 50 μm), com-
pared to our study (~ 40μm), which might be due to dif-
ferent growth conditions.
Under the conditions we used the average planar
area of bundle sheath chloroplasts in wild-type and
pZmUbi::ZmG2 was higher than in previous analysis
[22]. ese dierences might result from dierent experi-
mental conditions such as light intensity and/or from the
use of confocal laser microscopy to study chloroplasts in
our study. For example, Wang et al., 2017 used the sin-
gle-cell isolation method followed by bright-eld light
microscopy. ere is a possibility that chloroplast area is
over-estimated from chlorophyll autouorescence due to
the introduction of background pixels. To investigate this,
we measured the planar area of 574 bundle sheath chlo-
roplasts from the two-dimensional images of wild-type
rice leaf samples obtained from serial block-face scan-
ning electron microscopy (Additional le 3a). Based on
Fig. 6 Linear regression analysis between bundle sheath chloroplast
parameters and paradermal cell area of wild-type rice. Linear regression
plots representing bundle sheath (BS) paradermal cell area versus average
chloroplast area (a), maximum chloroplast area (b), chloroplast numbers
(c), total chloroplast occupancy (d), and relative chloroplast area in the cell
(%). Graphs show the line of best fit and standard error (grey filled region)
of the linear model fitted to the data (red circles)
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Billakurthi and Hibberd Plant Methods (2023) 19:69
this approach, the average area of individual chloroplasts
was 14 µm2 (Additional le 3b), which is higher than pre-
viously reported (11 µm2; [22]) but lower than what we
estimated from confocal imaging after ablation (16–17
µm2; Figs.4b and 5c). e reduction in bundle cell width
and chloroplast area from serial block-face scanning elec-
tron microscopy compared with confocal imaging data
might be due to tissue shrinkage during sample prepara-
tion [31, 32]. us, it implies that the larger chloroplast
area might result from our experimental conditions than
confocal imaging. Irrespective of these dierences, leaf
ablation in association with confocal imaging allows dif-
ferences between genotypes to be detected (Fig.4b, d).
Although, we measured only planar chloroplast area, rice
chloroplasts are often lobed [33, 34] and so in the future
being able to estimate volume and surface area will help
rene our understanding of the relationship between
photosynthesis activity and leaf anatomy.
Conclusions
In conclusion, we report a simple and scalable leaf abla-
tion method to access bundle sheath cell les in C3
species such as rice. We show that this method is appro-
priate to measure bundle sheath cell dimensions, chlo-
roplast areas and chloroplast numbers per cell. We also
show bundle sheath cells are intact after the leaf ablation.
As the approach is at least ten times faster than the next
most ecient approach, ablation should signicantly
accelerate analysis of transgenic lines harbouring candi-
date genes aimed at modifying the rice bundle sheath.
Materials and methods
Plant material and growth conditions
Seeds of wild-type (Oryza sativa spp japonica cv. Kita-
ake) and maize GOLDEN-2 (ZmG2) overexpressing rice
([22]; ZmUBIpro::ZmG2 line E131) were imbibed in ster-
ile Milli-Q water and incubated at 30°C in the dark for
two days. Seeds were transferred onto Petri plates with
moistened Whatman lter paper and germinated in the
growth cabinet at 28°C with 16/8 hrs. of light/dark cycle.
After two days, germinated seedlings were potted into 9
by 9cm pots (two plants/pot) lled with Prole Field and
Fairway soil amendment (www.rigbytaylor.com). Plants
were grown in a walk-in plant growth chamber under
a 12-hour photoperiod at a photon ux density of 400
µmol m-2s-1 at 28°c (day) and 20°C night. Once a week,
plants were fed with the Peters Excel Cal-Mag Grower
fertiliser solution (LBS Horticulture, Clone, UK) with
additionally supplied iron (Fe7 EDDHA regular, Garden-
ing Direct, UK). e working fertiliser solution contains
0.33g/L of Peters Excel Cal-Mag Grower and 0.065g/L
chelated iron. Maize (B73) seeds were germinated on wet
lter paper in the dark at 28°C for three days after which
each germinated seed was transferred into a two litre pot
containing a mixture of two parts nutrient-rich compost
(Levington Advance M3, ICL, Ipswich, UK) to one part
topsoil (Westland, Dungannon, Northern Ireland), 10 ml
Miracle-Gro all-purpose fertiliser beads and 15 ml Mira-
cle-Gro magnesium salt (Scotts Miracle-Gro, Marysville,
OH, USA). ey were grown in a growth cabinet operat-
ing at 28°C (day)/ 20°C (night) at a photon ux density
of 550 µmol m-2s-1 under a 14-hour photoperiod.
Sample preparation
e middle region of the fully expanded fourth leaf from
wild-type Kitaake, ZmUBIpro::ZmG2 overexpressing rice
lines and maize was xed with 1% (w/v) glutaraldehyde
in 1X PBS buer. Once xative was inltrated, samples
were left in that solution for about two hours and then
washed twice with 1X PBS buer, with each wash last-
ing ~ 30min. Leaf samples can be stored in 1X PBS buf-
fer at 4°C for several weeks without losing chlorophyll
autouorescence. Before microscopy, the adaxial side
of the xed leaf material was ablated gently with a ne
razor blade (Personna, Verona, VA 24,482; Additional
le 1) to remove mesophyll layers. is process requires
two to three minutes to scrape o the epidermis and
mesophyll tissue to expose rice bundle sheath cells sur-
rounding intermediate veins (Additional le 1). As maize
contains fewer mesophyll layers, it took less than a min-
ute to ablate mesophyll layers. Bundle sheath cells can
be directly visualized with light microscopy. For confo-
cal microscopy, the ablated leaf fragment was stained
with the cell wall stain calcouor white (0.1%; Sigma) for
5min and then rinsed twice with H2O.
Light and confocal laser microscopy
Light microscopy images (Olympus BX51 microscope) of
both rice and maize leaves were captured using an MP3.3-
RTV-R-CLR-10-C MicroPublisher camera and QCapture
Pro 7 software (Teledyne Photometrics, Birmingham,
UK) to visualize the dierences before and after the abla-
tion. A Leica SP8X confocal microscope upright system
(Leica Microsystems) was used for uorescence imaging.
It has two continuous wave laser lines, 405 and 442nm,
a 460–670nm super continuum white light laser (WLL)
and four hybrid detectors and one photomultiplier tube.
Imaging was conducted using a 25X water immersion
objective and Leica Application Suite X (LAS X; ver-
sion: 3.5.7.23225) software. Calcouor white was excited
at 405nm and emitted uorescence captured from 452
to 472nm. Chlorophyll autouorescence was excited at
488nm and emission captured 672–692nm. ree repli-
cates from both wild-type Kitaake and ZmUBIpro::ZmG2
overexpression line E131 were analysed. Z-stacks of ~ 30
lateral bundle sheath cells surrounding three dierent
intermediate veins (3°) and eight to ten cells per vein
were obtained from each replicate. From three replicates,
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 9 of 11
Billakurthi and Hibberd Plant Methods (2023) 19:69
82 and 90 bundle sheath cells from wild-type and E131
line were imaged respectively. Maximum intensity pro-
jection images were used to quantify bundle sheath cell
dimensions, individual chloroplast areas and chloroplast
number per cells. Bundle sheath cell length and width
were measured at the mid-point of the proximal-distal
and medio-lateral axes respectively. Images of 90 maize
bundle sheath cells of intermediate veins from three rep-
licates were captured using confocal laser microscopy to
measure bundle sheath cell dimensions.
Serial block-face scanning electron microscopy
Wild-type rice leaf (middle region of fourth leaves) sam-
ples were xed in xative (2% w/v glutaraldehyde / 2%
w/v formaldehyde in 0.05M sodium cacodylate buer pH
7.4 containing 2 mM calcium chloride) overnight at 4oC.
After washing ve times with 0.05 M sodium cacodyl-
ate buer pH 7.4, samples were osmicated (1% osmium
tetroxide, 1.5% potassium ferricyanide, 0.05 M sodium
cacodylate buer pH 7.4) for three days at 4oC. After
washing ve times in DIW (deionised water) samples
were treated with 0.1% (w/v) thiocarbohydrazide/DIW
for 20min at room temperature in the dark. After wash-
ing ve times in DIW, samples were osmicated a second
time for one hour at RT (2% osmium tetroxide/DIW).
After washing ve times in DIW, samples were block
stained with uranyl acetate (2% uranyl acetate in 0.05M
maleate buer pH 5.5) for three days at 4oC. Samples
were washed ve times in DIW and then dehydrated in
a graded series of ethanol (50%/70%/95%/100%/100%
dry), 100% dry acetone and 100% dry acetonitrile, three
times in each for at least ve minutes. Samples were inl-
trated with a 50/50 mixture of 100% dry acetonitrile/
Quetol resin mix (without BDMA) overnight, followed
by three days in 100% Quetol (without BDMA). en, the
sample was inltrated for ve days in 100% Quetol resin
with BDMA, exchanging the resin each day. e Quetol
resin mixture is: 12g Quetol 651, 15.7g NSA (nonenyl
succinic anhydride), 5.7g MNA (methyl nadic anhydride)
and 0.5g BDMA (benzyldimethylamine; all from TAAB).
Samples were placed in embedding moulds and cured at
60oC for three days.
Sections were cut at a thickness of about 70nm using
a Leica Ultracut E, placed on a Melinex plastic cover-
slip, and allowed to air dry. Coverslips were mounted on
aluminium scanning electron microscopy stubs using
conductive carbon tabs and the edges of the slides were
painted with conductive silver paint. en, samples were
sputter coated with 30nm carbon using a Quorum Q150
TE carbon coater. Samples were imaged in a Verios 460
scanning electron microscope (FEI/ermosher) at
4 keV accelerating voltage and 0.2 nA probe current
in backscatter mode using the concentric backscatter
detector (CBS) in eld-free mode for low magnication
imaging and in immersion mode at a working distance of
3.5-4mm; 1536 × 1024 pixel resolution, 3 us dwell time,
4 line integrations for higher magnication imaging.
Stitched maps were acquired using FEI MAPS automated
acquisition software using the default stitching prole
and 5% image overlap. Transverse bundle sheath cell
width was measured from bundle sheath cells of three
minor veins per replicate, and three biological replicates
were used. In total, dimensions of 92 bundle sheath cells
were measured. e planar chloroplast areas were mea-
sured from paradermal sections of bundle sheath cells
surrounding two minor veins per replicate. Total areas of
574 chloroplasts were measured across 130 cells.
Data analyses
Bundle sheath cell dimensions (length, width, and area),
chloroplast area and numbers per cell were measured
using ImageJ version 2.1.0/1.53c [35]. RStudio (ver-
sion:1.4.1106) was used to plot the data using the ggplot2
software package [36] and statistical analysis was per-
formed using the ggpubr software package [37]. First,
equality of variance between the two groups was tested
using Barlett’s test [38]. Where the assumption of equal
variance was met, a two-tailed pairwise t-test (Student’s
t-test) was performed. Otherwise, Welch’s two-sample
t-test was performed. A general linear regression model
was performed using the ggfortify package [39] and
assumptions of a linear regression model were tested
using the autoplot function of the ggfortify package.
Finally, the general linear regression line was tted using
the lm function and, ANOVA test was performed to test
whether the slope is signicantly dierent from zero.
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s13007-023-01041-x.
Additional le 1: Movie showing rice leaf ablation. Rice leaf image with
different vein orders before and after leaf ablation (top) and video show-
ing the leaf ablation process (bottom). A drop of water was added onto a
glass plate to prevent the dehydration while ablating the leaf. 1°: primary/
mid vein; 2°: secondary/large lateral veins; 3°: tertiary/intermediate veins.
Movie courtesy: Dr Satish Kumar Eeda. Additional le 2: Comparison of
bundle cell width in paradermal versus transverse sections obtained from
confocal laser scanning microscopy versus serial block-face scanning elec-
tron microscopy (SBF-SEM), respectively. (a) Transverse section of a rice leaf
obtained from serial block-face scanning electron microscopy, represent-
ing the bundle sheath cells of a tertiary vein (3°). Bundle sheath cell width
was measured at the mid-point of the medio-lateral axes as annotated
with a red arrow. (b) Comparison of bundle sheath cell width measure-
ments from paradermal and transverse sections, obtained from confocal
imaging (rice data from Fig. 3c) and serial block-face scanning electron mi-
croscopy, respectively. BS: Bundle sheath cell; M: Mesophyll cell. Blue dot
in the violin plots represent mean values. Statistical test: t-test. Additional
le 3: Comparison of individual chloroplast areas obtained from confocal
laser scanning microscopy versus serial block-face scanning electron
microscopy (SBF-SEM). (a) Paradermal section of a rice leaf obtained from
serial block-face scanning electron microscopy, representing the lateral
bundle sheath cells of a tertiary vein (3°). Bundle sheath chloroplasts were
pointed with red arrows. (b) Comparison of individual chloroplast areas
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 10 of 11
Billakurthi and Hibberd Plant Methods (2023) 19:69
from confocal (wild-type rice data from Fig. 4b) and two-dimensional
serial block-face scanning electron microscopy imaging. BS: Bundle sheath
cell; M: Mesophyll cell. Blue dot in the violin plots represent mean values.
Statistical test: t-test.
Acknowledgements
This research was funded by a C4 Rice Project grant (#INV-002970) from The
Bill & Melinda Gates Foundation to the University of Oxford. For the purposes
of open access, the authors have applied a Creative Commons Attribution
(CC BY) license to any Author Accepted Manuscript version arising from
this submission. We thank Dr Lei Hua for useful suggestions for confocal
laser microscopy work, Dr Lee Cackett for providing maize leaf material and
Dr Tina B. Schreier for her guidance on serial block-face scanning electron
microscopy work. We also thank Prof. Jane Langdale for providing rice seeds of
ZmUBIpro::ZmG2 overexpressing line. We thank Dr Karin H Müller and Georgina
E Lindop from the Cambridge Advanced Imaging Centre (CAIC) for the
electron microscopy sample preparation as well as image acquisition.
Author contributions
KB designed and performed the experiments. KB drafted the manuscript. KB
and JMH reviewed and edited the manuscript.
Data Availability
All data supporting the findings of this study are available within the paper
and within its supporting information data published online.
Declarations
Ethics approval and consent to participate
.
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 31 March 2023 / Accepted: 19 June 2023
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