Oxime-derived palladacycle Immobilized in an Ionic Liquid Brush as an Efficient and Reusable Catalyst for Mozoroki-Heck Reaction in Neat Water

Article abstract of DOI:10.1002/aoc.5226

An efficient and reusable heterogeneous catalyst with oxime-derived palladacycle immobilized in an ionic liquid brush has been synthesized and an environmentally-friendly procedure have been developed for coupling aryl iodides and bromides with acrylic acid. These reactions were conducted in neat water under aerobic conditions with water-insoluble or even solid aryl halides and they proceeded smoothly and cleanly without any organic co-solvent or other additives. The ionic liquid brush could be easily recovered and reused at least five times without significant loss of activity. The protocol has the advantages of excellent yields, environmental friendliness, and catalyst recyclability.

Full text of DOI:10.1002/aoc.5226

DR SHAWN MANSFIELD (Orcid ID : 0000-0002-0175-554X)
Article type : Original Article
The Class II KNOX genes KNAT3 and KNAT7 work cooperatively to influence
secondary cell wall deposition and provide mechanical support to Arabidopsis stems
Shumin Wang1, Masatoshi Yamaguchi2, Etienne Grienenberger3, Patrick T. Martone1, A. Lacey
Samuels1 and Shawn D. Mansfield4*
1. Department of Botany, University of British Columbia, Vancouver, BC, Canada
2. Graduate School of Science and Engineering, Saitama University, Saitama, Japan
3. Institut de biologie moléculaire des plantes (IPMB), CNRS UPR 2357, Strasbourg, France
4. Department of Wood Science, University of British Columbia, Vancouver, BC, Canada
* Corresponding Author
Shawn Mansfield
Shawn.mansfield@ubc.ca
Phone: 604-822-0196
KEYWORDS
This article has been accepted for publication and undergone full peer review but has not been through the
copyediting, typesetting, pagination and proofreading process, which may lead to differences between this
version and the Version of Record. Please cite this article as doi: 10.1111/TPJ.14541
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Knotted-like homeobox, KNAT3, KNAT7, stem biomechanics, interfascicular fiber, xylem vessel,
secondary cell wall, cellulose, lignin
SUMMARY
The transcription factor KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) is a Class II KNOTTED1-like
homeobox (KNOX2) gene that, in interfascicular fibers, acts as a negative regulator of secondary cell
wall biosynthesis. In addition, knat7 loss-of-function mutants display an irregular xylem (irx)
phenotype, suggesting a potential positive regulatory role in xylem vessel secondary cell wall
deposition. Although our understanding of the role of KNAT7 is evolving, the functions of the closely
related KNOX2 genes, KNAT3, KNAT4 and KNAT5, in secondary wall formation still remain unclear.
We found that all four Arabidopsis KNOX2 genes were expressed in the inflorescence stems,
however, only the knat3 knat7 double mutants showed a phenotype, displaying an enhanced irx
phenotypes relative to the single mutants, as well as decreased interfascicular fiber cell wall
thickness. Moreover, knat3 knat7 double mutants had reduced stem tensile and flexural strength
compared with wild-type and single mutants. In contrast, KNAT3 overexpression resulted in thicker
interfascicular fiber secondary cell walls in inflorescence stems, suggesting a potential positive
regulation in interfascicular fiber secondary wall development. This work identifies KNAT3 as a
potential transcriptional activator working together with KNAT7 to promote secondary cell wall
biosynthesis in xylem vessels, while concurrently acting antagonistically with KNAT7 to influence
secondary wall formation in interfascicular fibers.
INTRODUCTION
Owing to the presence of secondary cell walls, trees and other plants form the foundations of
terrestrial ecosystems. Primary and secondary cell walls are the two major structures surrounding
almost all plant cells. The newly formed wall is first defined by the primary cell wall, which is thin,
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expandable, and has a relatively constant proportion of three main cell wall polysaccharide-derived
polymers: cellulose, hemicelluloses, and pectin, as well as a minor amount of protein. In specialized
cells, when the primary wall has finished expanding, a secondary cell wall is deposited, which is
thicker and composed primarily of cellulose, hemicelluloses and lignin. Secondary cell walls confer
the main structural components of mature tracheary elements and fibers, providing additional
mechanical strength and stiffness to plant tissues (Cosgrove and Jarvis, 2012).
In land plants, the formation of secondary cell walls requires a complex transcriptional network,
which includes at least three layers of regulation (reviewed in Nakano et al., 2015; Yang and Wang,
2016). The VASCULAR-RELATED NAC-DOMAIN gene family (VND1-7) have been identified as key
regulators of secondary cell wall deposition in vessel elements (Kubo et al., 2005), while the NAC
SECONDARY WALL THICKENING PROMOTING FACTOR gene family (NST1/3) regulate secondary cell
wall deposition in interfascicular fibers (Zhong et al., 2006; Mitsuda et al., 2007). The second-layer
master switches are MYB-type transcription factors, such as MYB46 and MYB83, which are directly
regulated by the NAC transcription factors, and act as regulators of secondary wall formation in both
xylem vessels and fibers (Zhong et al., 2007; McCarthy et al., 2009; Ohashi-Ito et al., 2010; Wang et
al., 2011; Zhong and Ye, 2012). Many other transcription factors also function downstream of the
NAC and MYB domain master switches, forming a feed forward regulatory network for secondary
wall deposition (Zhong et al., 2008; Ko et al., 2009; Taylor-Teeples et al., 2014).
One of the Arabidopsis Knotted-like homeobox (KNOX) genes, KNOTTED ARABIDOPSIS THALIANA7
(KNAT7), has been shown to be a direct target of both SND1 (Zhong et al., 2008) and MYB46 (Ko et
al., 2009). Unlike the NAC and MYB domain transcription factors that activate secondary cell wall
deposition, KNAT7 was reported to act as a negative regulator of secondary cell wall formation in
interfascicular fibers, with thicker fiber walls observed in loss-of-function KNAT7 mutants (Li et al.,
2012). Paradoxically, Zhong et al. (2008) found that transgenic plants with dominant repression of
KNAT7 had a reduction in secondary wall thickness. Additionally, knat7 mutants had an irregular
xylem (irx) phenotype (Li et al., 2012), as reported previously in the original irregular xylem studies
(Brown et al., 2005). Recently, He et al. (2018) also discovered that KNAT7 positively impacts xylan
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biosynthesis. Based on the identification of KNAT7 interactors, such as OVATE FAMILY PROTEIN4
(OFP4), BELL-LIKE HOMEODOMAIN6 (BLH6), and MYB75 (Hackbusch et al., 2005; Bhargava et al.,
2010; Li et al., 2011; Liu et al., 2014), KNAT7 may display defined spatiotemporal functions in
secondary wall formation, depending on specific cell type and interacting partners. However, the
specificities of the mechanism of KNAT7 regulation of secondary cell wall biosynthesis are still poorly
understood.
Along with KNAT7, there are in total nine KNOX genes in Arabidopsis, which belong to the plant-
specific THREE AMINO ACID LOOP EXTENSION (TALE) homeodomain superfamily (Hake et al., 2004;
Hay and Tsiantis, 2010). Based on gene duplication and diversification events in a common ancestor
of land plants, KNOX genes were grouped into two classes; class I (KNOX1) and class II (KNOX2;
Kerstetter et al., 1994; Mukherjee et al., 2009). A new class of KNOX genes, KNATM, which does not
have a homeodomain, has been shown to regulate the leaf proximal-distal patterning and has only
been identified in dicot species thus far (Kimura et al., 2008; Magnani and Hake, 2008). KNOX1 genes
include SHOOTMERISTEMLESS (STM), BREVIPEDICELLUS (BP), KNAT2 and KNAT6, which are involved
in cell proliferation (Sakakibara et al., 2008) and required for development of shoot apical meristem
(Long et al., 1996; Vollbrecht et al., 2000; Groover et al., 2006; Tsuda et al., 2011). KNOX2 genes
(KNAT3, KNAT4, and KNAT5) have been shown to function redundantly to influence lateral organ
differentiation in Arabidopsis (Furumizu et al., 2015). KNAT3 was also reported to modulate abscisic
acid (ABA) responses during germination and early seedling development (Kim et al., 2013). KNAT7
belongs to the KNOX2 clade, and since it plays diverse roles in secondary wall formation, the
question arises whether the other three KNOX2 genes, KNAT3, KNAT4 and KNAT5, may also play a
role in secondary wall formation.
Here, we report the secondary cell wall characteristics of knat3, knat4 and knat5 mutants in
Arabidopsis stems. We found that all KNOX2 genes are expressed in the inflorescence stems, and
loss-of-function analysis show knat3 knat7 double mutants exhibit an enhanced irregular xylem (irx)
phenotype, typified by weak inflorescence stems. In addition, the interfascicular fiber wall thickness
is reduced and cell wall compositions are modified in knat3 knat7 double mutants. Constitutive
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overexpression of KNAT3 led to thicker interfascicular fiber walls. Whole-genome expression
profiling of knat3 knat7 double mutants showed differential gene expression associated with cell
wall and secondary metabolism genes. Our results suggest that KNAT3 plays a positive role in
interfascicular fiber secondary wall formation, and may work cooperatively with KNAT7 to
contribute to cell wall deposition and the integrity of the cell-wall matrix, ultimately affecting plant
stem strength. Together, the KNOX2 proteins appear to form a complex network of positive and
negative regulators of secondary cell wall formation.
RESULTS
KNOX2 genes are expressed in the inflorescence stems
In Arabidopsis, there are four genes encoding KNOX2 proteins, KNAT3, KNAT4, KNAT5 and KNAT7
(Furumizu et al., 2015). To examine if the KNOX2 genes are expressed in cells undergoing secondary
wall deposition, and overlapping with that of KNAT7, Arabidopsis lines containing ProKNAT3::GUS,
ProKNAT4::GUS, ProKNAT5::GUS, and ProKNAT7::GUS were generated, and similar stem expression
patterns were observed among all independent transformants (Figure 1). All putative KNOX2
promoters directed GUS expression to the cortical cells and developing vascular bundles near the
apex of the stems (Figure 1a-d). This expression pattern was retained in the more mature stem
regions, where GUS staining was also detected in the differentiating interfascicular fibers (Figure 1e-
h). Interestingly, the expression of all four KNOX2 genes was absent in fully differentiated
interfascicular fibers and metaxylem vessels in the lower regions of mature inflorescence stems, but
was still apparent in cortical cells, phloem, and protoxylem cells (Figure 1i-l). These findings show
that KNAT3, KNAT4 and KNAT5 share similar expression patterns with KNAT7 in the different
developmental stages of inflorescence stems, and they are expressed in differentiating
interfascicular fibers and xylem cells where secondary cell walls are actively being deposited.
knat3 knat7 double knock-out mutants have enhanced irregular xylem (irx) phenotype
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To examine the role of KNOX2 genes in xylem development, their null alleles were obtained, which
included SALK_136464 (knat3), SALK_020216 (knat4-1), N759461 (knat4-2), SALK_000339C (knat5),
and SALK_002098 (knat7) (Kim et al., 2013; Furumizu et al., 2015; Li et al., 2012). Stem cross-
sections of each genotype were assessed at the base of mature inflorescence stems to determine
xylem morphology. knat3, knat4, and knat5 single mutants exhibited normal vessels morphology
(Figure 2b-d). As previously reported (Brown et al., 2005; Li et al., 2012; Liu et al., 2014), knat7 single
mutant exhibited an irregular xylem (irx) phenotype (Figure 2e). Due to the significant sequence
similarity and overlapping expression profiles of the KNOX2 genes, functional redundancy may
potentially mask the effects of single mutants loss-of-function on xylem and fiber cells. To test this
hypothesis, all possible combinations of double and triple mutants were generated (knat3 knat7,
knat4-1 knat7, knat5 knat7, knat3 knat4-1 knat7, knat3 knat5 knat7) or generously obtained from
Dr. John Bowman (knat3 knat4-2, knat3 knat5, knat4-2 knat5, knat3 knat4-2 knat5). Transverse
sections isolated from basal inflorescence stems of the mutants were stained histochemically with
toluidine blue and phloroglucinol (Figure S1).
Interestingly, the xylem vessels of the knat3 knat7 double mutant were more frequently irregular
and collapsed compared to wild-type and the corresponding single mutants (Figure 2f; Figure S1).
The enhanced irx phenotype of knat3 knat7 was obvious in the vascular bundles of all plants
examined. In contrast, knat4-1 knat7 and knat5 knat7 showed a mild irx phenotype (Figure S1),
which appeared to vary in severity between vascular bundles and plant lines. This “mild” irx
phenotype was similar to that commonly observed in knat7 single mutants (Figure 2e; Figure S1; Liu
et al., 2014). The other double mutants did not show any obvious change in xylem morphology
compared to wild-type plants (Figure S1). Cross-sections of triple mutant plants clearly showed that
knat3 knat4-1 knat7 and knat3 knat5 knat7 had severe irx phenotypes, mimicking that of knat3
knat7 double mutants, while knat3 knat4 knat5 vessels appeared similar to that of wild-type plants
(Figure S1). These results indicate that in addition to KNAT7, KNAT3 also plays a role in the xylem
vessel development. Based on our current findings, it would appear that KNAT3 and KNAT7 have
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overlapping functions in xylem development, and since the phenotype is more severe in the double
mutant, they are only partially functionally redundant.
Loss of KNAT3 and KNAT7 functions affects stem mechanical properties and fiber wall thickness
Phenotypic analysis of 7-week-old Arabidopsis plants revealed a striking impairment in upright
stature in knat3 knat7 double mutants compared to wild-type plants (Figure 3a, S2), suggesting
altered stems structure. To examine this phenotype more thoroughly, both uniaxial tensile tests and
three-point flexural tests of stems were performed. As described previously (Turner, 1997;
MacMillan et al., 2010), Arabidopsis stem strength is estimated as the maximum stress at yield for
breaking, and the stem stiffness (as defined by modulus of elasticity) is a measure of the force
required to deform the stem. Consistently, these stem integrity analyses showed that the bases of
knat3 knat7 fresh stems were considerably weaker than wild-type plants, displaying an
approximately 80% and 60% reduction in the tensile strength and tensile stiffness, respectively
compared to wild type (Figure 3b,c). The reduction in flexural stiffness in knat3 knat7 stem bases
was particularly remarkable in dried stems (82%, n=5-10), whereas fresh stems showed only a 56%
reduction compared to wild-type plants (Figure 3d), as the residual water in fresh stems help
maintain the cell turgidity, and as a consequence the variation (compared to wild-type plants) is not
as great.
Arabidopsis interfascicular fibers impart the necessary mechanical strength to inflorescence stems,
as has been demonstrated in a number of mutants, including ifl1 and fra8 (Zhong, 1997; Zhong,
2005), to mention only a few. To determine whether the pendent stem phenotype in the knat3
knat7 double mutants was caused by defects in interfascicular fibers, the fiber wall thickness was
examined. Cross-sections of stem bases stained with phloroglucinol-HCL clearly showed that the
interfascicular fiber wall thickness of knat3 knat7 double mutant was thinner (decreased by 37%,
n=30-50; Figure 4e) than that of wild type (Figure 4a-d), as well as knat3 and knat7 single mutant
plants. In contrast, as has previously been reported (Li et al., 2011; Li et al., 2012; Liu et al., 2014)
knat7 had significantly increased interfascicular fiber wall thickness (Figure 4e).
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KNAT3 and KNAT7 expression affects secondary cell wall composition
To further examine the role of these genes, individually and in combination, we investigated the
impact of these knockouts on secondary cell wall composition on the basal segments of
inflorescence stems. While single mutants had no significant (mild) changes in the concentrations of
sugars derived from cell wall carbohydrates, the double mutant knat3 knat7 showed substantial
compositional changes (Table 1). The glucose, xylose and mannose content in the double mutants
was significantly reduced (22%, 43% and 40%, respectively), while considerable increases were
observed in arabinose, rhamnose and galactose, displaying a 75%, 41% and 41% increase
respectively, compared to wild-type plants (Table 1). Given cellulose is a polymer of (1,4)-linked
glucose, the decreased total glucose content could be attributed to a reduction in stem cellulose
deposition, however, it could also be from hemicellulose-derived polymers. To test the hypothesis,
α-cellulose content was quantified. Indeed, the double knat3 knat7 mutants showed an approximate
20% reduction in cellulose content compared to the wild type, consistent with the observed
reduction in total cell wall glucose content (Table 2). There was no discernible change in the (acid-
insoluble and soluble) lignin content of knat3 single mutant relative to the wild-type control plants
(Table 2). In contrast, knat7 had an 11% increase in acid-insoluble lignin and 19% decrease in soluble
lignin, while knat3 knat7 displayed a 4% decrease in acid-insoluble lignin and a 26% increase in
soluble lignin (Table 2). This observation is interesting, as the current observation and those
previously reports (Li et al., 2012; Liu et al., 2014, He et al., 2018) conclusively show that the knat7
single mutant had increased total lignin, while the knat3 knat7 double knockout mutant
unexpectedly did not show a change in total lignin (Table 2).
In addition to total lignin content, lignin monomer composition was assessed by thioacidolysis. knat3
and knat7 single mutants had lower S/G lignin ratio than the corresponding wild-type control plants,
while the ratio was strikingly lower in the knat3 knat7 double mutant stems (84% reduction
compared to the wild type; Figure 5a). Mäule staining of stem cross-sections was consistent with the
chemical analysis (Figure 5b), clearly showing that the vascular elements and interfascicular fibers
displayed the classical red coloration in wild-type and single mutants plants, which is indicative of
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syringyl-derived lignin, while a drastic reduction in red coloration was observed in the knat3 knat7
double mutants (Figure 5b).
knat3 knat7 phenotypes are complemented by ProKNAT3::KNAT3-GFP and ProKNAT7::KNAT7-GFP
To investigate whether the knat3 knat7 phenotypes were caused by loss of function of KNAT3 and
KNAT7, complementation experiments were performed. knat3 knat7 was transformed with two
different constructs, ProKNAT3::KNAT3-GFP and ProKNAT7::KNAT7-GFP. Expression of KNAT3 under the
control of its native promoter partially rescued the severe irx phenotype consistently observed in
knat3 knat7, manifesting in a phenotype similar to knat7 (Figure 6a). Plants transformed with
ProKNAT7::KNAT7-GFP in the knat3 knat7 background fully rescued the irx phenotypes of knat3 knat7
(Figure 6a). The pendent phenotype of knat3 knat7 was also complemented in both transformants
(Figure 6b). The interfascicular fiber wall thickness was quantified in inflorescence stems of the
complementation lines (Figure 6c,d), and shown to be restored to a level relative to the wild-type
plants by expressing KNAT3-GFP under the control of its native promoter in knat3 knat7 background
(Figure 6c), mimicking the knat7 single mutant. As with knat3 single mutants, the interfascicular
fiber cell wall thickness was not different from that of wild type when KNAT7-GFP was expressed in
knat3 knat7 mutants (Figure 6c).
KNAT3 and KNAT7 were also confirmed to have overlapping expression patterns during secondary
cell wall deposition, as reflected in the GFP signals apparent in the complemented lines, since both
lines were under the control of their native promoters (Figure S3). KNAT3 had a broader expression
pattern compared with KNAT7, but both were detected in the protoxylem and metaxylem vessel
precursors in the roots, and in the interfascicular fiber cells in inflorescence stems of the
complemented lines (Figure S3).
Overexpression of KNAT3 increases interfascicular fiber wall thickness of stems
The contrasting phenotypes of knat3 knat7 double mutants having decreased interfascicular fiber
wall thickness, and knat7 single mutants displaying increased fiber wall thickness, suggested that
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KNAT3, along with KNAT7, plays a regulatory role in the development of interfascicular fibers. In an
attempt to enhance the levels of KNAT3, plant lines overexpressing KNAT3 under the control of 35S
promoter were obtained from Dr. John Bowman (Furumizu et al., 2015). Cross-sections of the basal
inflorescence stems were examined by toluidine blue staining (Figure 7) and compared to wild-type
plants, KNAT3 overexpression lines appeared to have thicker interfascicular fiber secondary cell
walls (Figure 7a) while the those of the corresponding vessels appeared normal. Measurements
taken via high-magnification light microscopy images confirmed that constitutive expression of
KNAT3 (Pro35S::KNAT3) significantly increased interfascicular fiber wall thickness (Figure 7b),
suggesting that KNAT3 plays a role in the positive regulation secondary wall formation in
interfascicular fibers.
Genome-wide transcript profiling shows altered expression of secondary cell wall related genes in
knat3 knat7 plants
In an attempt to further elucidate the roles of KNAT3 and KNAT7 in secondary cell wall development,
the transcript abundance of known cellulose, hemicellulose and lignin biosynthetic genes was
profiled by qRT-PCR in cDNA isolated from basal stems of knat3, knat7 and knat3 knat7 mutants. It
was clearly shown that the expression levels of secondary cell wall specific CELLULOSE SYNTHASE
(CESA) genes (CESA4, CESA7, CESA8), and one of xylan biosynthetic gene (IRX9) were dramatically
increased in the double mutants compared with wild-type and the individual single mutants (Figure
8a). Another xylan biosynthetic gene, IRX10, was significantly down-regulated in knat7 and knat3
knat7 plants, while knat3 showed a slight increase in IRX10 expression (Figure 8a). Loss of KNAT3
and KNAT7 functions together also resulted in both up-regulation and down-regulation of several
phenylpropanoid biosynthetic genes (Figure 8b). Specifically, the expression of C4H, C3’H/REF8,
CCoAMT1 and CCR1 were elevated 1.5- to 2- fold in the basal segments of knat3 knat7 inflorescence
stems, while the abundance of FERULATE-5-HYDROXYLASE (F5H), a key enzyme for syringyl lignin
biosynthesis, was considerably decreased (approximately 50%) in the knat3 knat7 mutant (Figure
8b). This later finding is consistent with the reduced syringyl lignin observed in our biochemical and
histochemical assessment of the knat3 knat7 stems.
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Untargeted transcriptome analysis was also carried out by mRNA sequencing of inflorescence stems
to identify genes differentially expressed among wild-type, knat3, knat7 and knat3 knat7 stems (File
S1). Compared with wild type, knat3 knat7 had a total of 959 up-regulated and 1001 down-regulated
genes, whereas knat3 and knat7 had 484 and 367 down-regulated, respectively (Figure 9a,c).
Comparative analyses revealed that of all the mis-regulated genes, 32 and 117 genes were found in
common in knat3, knat7 and knat3 knat7 plants (Figure 9a,c). To determine the identities of these
differentially expressed genes (DEGs), GO ontology classifications was performed based on their
known functions. In knat7, genes involved in cell wall organizational processes were significantly up-
regulated and over-represented compared to wild-type plants (Figure 9b), which is consistent with a
repressor-type function in secondary cell wall formation (Li et al., 2012). The expression level of
KNAT3 was not affected in the knat7 mutant background (File S1). Compared with knat3 and knat7,
a significant number of genes related to glucuronoxylan biosynthesis were up-regulated in knat3
knat7, as well as plant-type secondary cell wall biogenesis, nucleotide-sugar biosynthetic processes,
microtubule-based processes and pectin metabolic processes (Figure 9b). Additionally, a few
biological processes were over-represented in the down-regulated DEGs found in knat3 knat7 plants,
such as aromatic amino acid family catabolic process, phloem transport, and response to red light,
to mention only a few (Figure 9d). MapMan was used to visualize the differentially expressed genes
in various metabolic pathways (Figure S4). Consistent with the qRT-PCR data, knat3 knat7 exhibited
both increased and decreased transcript levels of seconday cell wall-related biosynthetic genes
(Figure S4, File S2).
DISCUSSION
KNAT7, one of the four KNOX2 genes in Arabidopsis, has been proposed to function as either a
transcriptional repressor or activator regulating secondary cell wall formation in different cell types
(Li et al., 2011; Li et al., 2012; He et al., 2018). The three other Arabidopsis KNOX2 genes, KNAT3,
KNAT4, and KNAT5, were found to act redundantly in regulating plant development, with serrated
leaves apparent on knat3 knat4, knat3 knat5 and knat3 knat4 knat5 plants (Furumizu et al., 2015).
However, little is known about their exact biological roles, if any, in secondary cell wall formation. In
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this study, beyond the developmental roles of the KNOX2 family members in leaf development, we
focused on their putative roles in secondary cell wall formation in complement to KNAT7. KNAT3
was identified as a potential transcriptional activator, working antagonistically with KNAT7 to impact
secondary wall formation in interfascicular fibers, while in xylem vessels, KNAT3 and KNAT7 function
together to influence secondary cell wall deposition.
Expression data shows that all four KNOX2 genes have overlapping expression domains in the
inflorescence stem, notably in tissues with active secondary cell wall deposition (Figure 1 and S3),
suggesting KNAT3, KNAT4 and KNAT5 may also play a role in secondary cell wall formation.
Interestingly, ProKNAT3::KNAT3-GFP has an expanded expression pattern compared with
ProKNAT7::KNAT7-GFP which is apparent in the roots, showing expression throughout other root cell
layers, other than protoxylem and metaxylem vessel precursors (Figures S3). This alone implies that
KNAT3 may play multiple roles in plant development, which is consistent with previous findings
suggesting that KNAT3 can modulate ABA responses (Kim et al., 2013) and lateral organ
development (Furumizu et al., 2015).
The collapsed xylem vessel phenotype, irx, has been widely used to identify and isolate Arabidopsis
mutants defective in the secondary cell wall biosynthesis (Turner, 1997; Jones et al., 2001; Brown et
al., 2005). Analysis of knat4 knat7, knat5 knat7 mutants revealed an irx phenotype, which
phenocopied knat7, while knat3 knat7 afforded an enhanced irx phenotype (Figure 2, S1). The
synergistic effect of the xylem vessel phenotype in double mutants indicates that KNAT3 and KNAT7
function redundantly in modulating vessel secondary cell wall development, although KNAT7 likely
plays the dominant role, as the knat7 single mutant displays the irx phenotype, while knat3 alone
has no obvious xylem vessel phenotype (Figure 2).
A striking effect of loss-of-function KNAT3 and KNAT7 was the weak inflorescence stem with reduced
tensile and flexural properties (Figure 3). Thinner interfascicular fiber cell walls in the knat3 knat7
double mutants is undoubtedly a major contributor to the pendent stem phenotype, as
interfascicular fibers are important for the mechanical strength of Arabidopsis inflorescence stems
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(Zhong, 2005; Zhong et al., 2007). Although the thicker secondary cell walls phenotype in
interfascicular fibers in knat7 mutants was observed here, as well as in previous studies (Li et al.,
2012; He et al., 2018), the double mutants knat3 knat7 displayed an opposing trend with thinner
interfascicular fiber wall thickness (Figure 4). Thus, the thicker secondary cell wall phenotype in
knat7 appears to be dependent on a functional KNAT3, but it is not associated with KNAT3
expression levels, as we did not observe a change in the transcript abundance of KNAT3 in knat7
stems compared with wild type (File S1). The complementation tests in our study also confirmed the
involvement of KNAT3 and KNAT7 in the regulation of Arabidopsis secondary cell wall stems (Figure
6). KNAT3 activates secondary cell wall formation in fibers, as ectopic expression of KNAT3
manifested in thicker interfascicular fiber secondary cell walls (Figure 7). However, knat3 single
mutants do not show an obvious interfascicular fiber secondary cell wall development phenotype
(Figure 4), which indicates that KNAT3 has some redundancy with other genes involved in regulating
secondary wall formation.
KNAT7 may trigger different regulatory sequences by interacting with different partners in different
cell types, similar to the functions of helix-loop-helix leucine zipper domain (bHLHZip) transcription
factor family proteins demonstrated in animals (reviewed in Lüscher, 2001; Amoutzias et al., 2008).
MYB75, BLH6 and OFP4 have been identified as KNAT7 interactors contributing to negatively impact
secondary cell wall deposition in interfascicular fibers and lignin biosynthesis (Bhargava et al., 2010;
Li et al., 2011; Liu et al., 2014). Zhong et al. (2008) found dominant transcriptional repression of
KNAT7 caused reduction in secondary cell wall thickening in interfascicular fibers, which is similar to
the knat3 knat7 double mutant phenotype. Since the dominant repression approach may not only
inhibit the function of the targeted transcription factor, but also their homologs (Hiratsu et al., 2004),
it is possible that the thinner fibers phenotype emanates from the repression of both KNAT3 and
KNAT7. Transactivation analyses have revealed that KNAT7 can not only repress REVOLUTA
expression by interacting with BLH6, but also directly activate IRX9 in Arabidopsis stems (Liu et al.,
2014; He et al., 2018). This work confirms knat7 mutant phenotypes with thicker interfascicular
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fibers and collapsed xylem vessels, supporting the view that KNAT7 has different functions in
different cell types.
The secondary cell wall quality is significantly affected in knat3 knat7 mutants, indicating that
KNAT3/KNAT7 influence key components of secondary cell walls in inflorescence stems. It is well
known that polymeric cellulose is the major load bearing component affecting stem tensile and
flexural strength, as clearly demonstrated in Arabidopsis and rice mutants (Turner and Somerville,
1997; Li et al., 2003). The reduced cellulose content of knat3 knat7 stem cell walls (Table 2) is, along
with the overall thinner walls, contributing to the observed decrease stem strength properties of
knat3 knat7. In addition to the altered cellulose content, the lignin composition was also modified in
the knat3 knat7 double mutants, with significant reductions in both syringyl lignin and consequently
the S/G lignin ratio in inflorescence stems (Table 2). These data suggests that KNAT3/KNAT7 could
positively impact both cellulose and lignin monomer biosynthesis. The observation that total lignin
content in knat3 knat7 stems was not changed, unlike knat7 single mutants, points to a potential
antagonistic function for KNAT3 and KNAT7 in regulating total lignin biosynthesis. KNAT7 was
reported to be a transcriptional activator in xylan biosynthesis (He et al., 2018). The considerable
reduction in the xylose content of knat3 knat7 supports a role for KNAT3 and KNAT7 in activation of
xylan biosynthesis. The knat3 knat7 mutant analysis indicates that these transcription factors are
important in secondary cell wall component biosynthesis.
The transcriptional impact of KNAT3 and KNAT7 on the molecular underpinnings of cell wall
development was assessed by both qRT-PCR and mRNA sequencing. Both techniques clearly show
increased expression of the secondary cell wall-specific cellulose biosynthetic genes (CESA4, CESA7
and CESA8) in the basal stems of knat3 knat7 mutants (Figure 8), which was unexpected as the knat3
knat7 mutants had lower cellulose contents (Table 2). However, the RNA levels of cellulose
biosynthetic genes may not match directly with their final protein contents. We reasoned that the
observed up-regulation of cellulose synthase genes might reflect a feedback mechanism where
these specific mutants are attempting to over compensate for the decreased cellulose content in
knat3 knat7 stems. The reduced cellulose content in the double mutant plants may be due to loss of
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functional cellulose synthase complex or decreased production of UDP-glucose, as several cellulose-
related genes were mis-regulated, including the down-regulation of cellulose synthase-like (CSL)
genes, CSLG3 and CSLA9, while CSLC4, COBRA-LIKE4 (COBL4), and KOR were highly up-regulated (File
S2). The key enzyme for syringyl monomer biosynthesis, F5H, was significantly down-regulated in the
knat3 knat7 tissue (Figure 8), which is consistent with the observed reduction in both syringyl lignin
and the associated S/G lignin ratio (Figure 5). mRNA-seq experiments provided a near-exhaustive
overview of the effects of the various mutation on gene expression in the stem. The down-
regulation of F5H was again apparent in the transcriptome analysis, and it was one of the most
highly down-regulated genes in knat3 knat7 stems. In addition, a number of secondary cell wall-
related transcription factors and biosynthetic genes were only differentially expressed in the knat3
knat7 double mutants (File S1,S2). Among the down-regulated cell wall-related genes were MAP70-5,
which encodes a plant-specific microtubule-associated protein that has previously been shown to be
important for secondary cell wall patterning (Pesquet et al., 2010; Oda and Fukuda, 2012), VND-
INTERACTING2 (VIN2), which is a transcriptional repressor regulating xylem cell specification
(Yamaguchi et al., 2010), and PECTIN METHYLESTERASE35 (PME35), which has also been shown to
have a pendent stem phenotype and an increased deformation rate of stem in the loss-of-function
mutant (Hongo et al., 2012). GO classification analysis clearly showed the cell wall organization
process was overrepresented in knat7 DEGs (Figure 9b), which may explain the thicker
interfascicular fibers and higher lignin phenotypes in knat7 mutants. The mRNA-seq also highlighted
a number of up-regulated genes in the knat3 knat7 plants involved in secondary cell wall biogenesis
process, which is consistent with qRT-PCR results. The up-regulation of these genes in the knat3
knat7 mutant could represent a negative feedback mechanism for secondary cell wall development.
MAPMAN analysis revealed a number of cell wall and secondary metabolism-related genes that
were uniquely affected in the knat3 knat7 mutants (Figure S4, File S2), which is correlated with their
weaked stem traits and heighten irx phenotypes. It is interesting that most of reported genes (Hao
and Mohnen, 2014) that contribute to irx phenoytpes were highly expressed in the double mutants,
except IRX10 (File S1). However, the irx10 mutant only has a mild irx phenotype (Wu et al., 2009).
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These findings suggests that KNAT3 and KNAT7 are impacting secondary cell wall biosynthesis
through an alternative mechanism, which is different from that described in the known IRX genes.
Taken together, we found that KNOX2 genes play important roles in secondary cell wall formation in
Arabidopsis. They are partially redundant, but also exhibit distinct functions in different cell types.
The knat3 mutation could enhance knat7 xylem vessel and syringyl lignin secondary cell wall
phenotypes (Figure 2, 5), while concurrently repressing the knat7 interfascicular fiber phenotype
(Figure 4). Based on these results, KNAT3 is a potential activator of xylem vessel secondary cell wall
formation, acting together with KNAT7, while it acts antagonistically with KNAT7 for secondary wall
formation in interfascicular fibers. Our findings improve the understanding of secondary cell wall
transcriptional networks, showing KNAT3 and KNAT7, either directly or indirectly, influence
secondary cell wall biosynthetic genes.
EXPERIMENTAL PROCEDURES
Plant material and growth condition
Arabidopsis thaliana ecotype Columbia was used as wild-type plants in all experiments, and all the
transgenic lines and mutants are also in the Columbia background. T-DNA insertion lines for knat3
(SALK_136464), knat4-1 (SALK_020216) and knat5 (SALK_000339C) were obtained from the
Arabidopsis Biological Resource Center (ABRC). The knat7-1 allele described in Li et al. (2012) was
used for all knat7 phenotypic analyses. Homozygous T-DNA insertion lines were screened by PCR
using gene-specific primers (Table S1). knat3, knat4-1, knat5, and knat7 alleles were used to
generate knat3 knat7, knat4-1 knat7, knat5 knat7, knat3 knat4-1 knat7 and knat3 knat5 knat7
double and triple mutants. The other double and triple mutants, knat3 knat4-2, knat3 knat5, knat4-2
knat5 and knat3 knat4-2 knat5 were kindly gifted from Dr. John Bowman (Furumizu et al., 2015). For
the overexpression analysis of KNAT3, homozygous lines for 35S::KNAT3 (Furumizu et al., 2015) were
employed for all experiments.
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In all experiments, seeds were sterilized with 70% ethanol and sown on Murashige and Skoog (MS)
medium with 1% sucrose, then cold-treated at 4°C for 48 hours in the dark, and grown at 20°C under
a 16/8 h (light/dark) photoperiod at about 120 umol m-2 s-1 light for 7 to 10 days. Seedlings were
transferred to soil and grown under long-day conditions (16/8 h light/dark cycle) at ~100 umol m-2 s-1
light, 20°C in growth chambers for further analysis.
Cloning and plant transformation
To generate ProKNAT3::GUS, ProKNAT4::GUS, ProKNAT5::GUS, and ProKNAT7::GUS constructs, the fragment
upstream of the ATG start codons of KNAT3 (3207 bp), KNAT4 (3290 bp), KNAT5 (1119 bp) and
KNAT7 (2591 bp) respectively, were sub-cloned into the pCR8/GW/TOPO entry vectors, and then
subsequently cloned into the binary vector PMDC163. For complementation experiments, the
genomic fragments of KNAT3 and KNAT7 containing the 5’ promoter region and 3’ untranslated
regions were amplified from Col-0 genomic DNA, respectively. Following verification of the
nucleotide sequences of the amplified fragments, each fragment was cloned into the binary vector
PMDC107 containing the hygromycin resistance genes to generate the ProKNAT3::KNAT3-GFP and
ProKNAT7::KNAT7-GFP constructs. Gene-specific oligonucleotides used for cloning and construct
generation are shown in Table S1.
All the constructs were introduced into Agrobacterium tumefaciens strain GV3101 for plant
transformation. Wild-type Columbia was transformed for expression pattern analysis, while the
complementation constructs were transformed into knat3 knat7 double mutant lines via the floral
dip method (Clough and Bent, 1998) to generate transgenic plants.
GUS expression assay
GUS activity was assayed on hand-sections of 8-week-old inflorescence stems by incubating tissues
in a solution containing 0.1M sodium phosphate buffer (PH 7.0), 1mM substrate 5-bromo-4-chloro-
3-indolyl-β-D-glucuronide (X-Gluc), 0.5mM potassium ferricyanide and 0.01%(v/v) Triton X-100 at
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37°C for 1 h to overnight. The resulting stained tissues were fixed with FAA (50 [v/v] ethanol, 5%
[v/v] acetic acid and 10% [v/v] formaldehyde), and observed with an Olympus AX70 light microscope.
Microscopy
Freshly harvested 8-week-old inflorescence stems were hand-sectioned, stained with aqueous 0.05%
toluidine blue O for 1-2 min and mounted with water, or stained with 2% (v/v) phloroglucinol for 30
sec and mounted with concentrated HCl. Mäule staining was performed by treating hand sections
for 5 min with 0.5% KMnO4, rinsing in water, and treating with 30% HCl until the brown colour
disappeared. Samples were mounted in concentrated NH4OH, and viewed using an Olympus AX70
light microscope. ImageJ was used to measure the thickness of interfascicular fibers. For each cell,
three measurements were taken, and the average used as the value of that cell. Statistical analysis
was performed by Student’s T-test and one-way ANOVA followed by Tukey’s post hoc test.
Roots of ProKNAT3::KNAT3-GFP and ProKNAT7::KNAT7-GFP plants in the knat3 knat7 background were
counterstained with propidium iodide and observed on a Perkin-Elmer UltraView VoX spinning disk
confocal mounted on a Leica DMI6000 inverted microscope with a Hamamatsu 9100-02 CCD camera.
The microscope used for all live-cell imaging was fit with the following excitation/emission filters:
GFP (488 and 525) and propidium iodide (620/720). ImageJ software was used for image processing.
Physical tests
Tensile and three-point flexural tests were carried out using a 5565 model Instron universal testing
machine according to the detailed methods describe by MacMillan et al. (2010). 60mm basal stem
samples were harvested and used for all tensile tests. The 20mm basal segments were attached to
paper tabs by moisture-activated cyanoacrylated Loctite 454 gel (Henkel, Dusseldorf, Germany). The
instron testing machine was fit with a 5kN load cell, and employed a cross-head speed of 10
mm/min. Flexural bending strength was estimated on a specimen 15mm from the stem base, using
the Instron fitted with a 10N load cell and a cross head speed of 10mm/min. A digital caliper was
used to measure the diameter of each stem. BlueHill software was employed to capture and
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calculate the force and modulus of elasticity for each sample. Statistical analysis was performed by
one-way ANOVA followed by Tukey’s post hoc test.
Cell-wall analysis
All the cell-wall analyses were performed on dried stems harvested 0-11cm from the base. Lignin
content was determined by the modified Klason method described previously (Coleman et al., 2008),
in which 100mg of ground, hot acetone-extracted Arabidopsis stem tissue was incubated with 72%
H2SO4 for 2 hours with regular stirring. Then, the reaction was diluted to 3% H2SO4 and autoclaved
to completely hydrolyze cell wall polysaccharides. The monosaccharides were quantified by HPLC
(DX-500; Dionex) equipped with a CarboPac PA1 column and a pulsed amperometric detector with a
gold electrode. Acid-insoluble lignin was quantified gravimetrically, while the acid-soluble lignin
content was measured spectrophotometrically at 205 nm.
-cellulose content was estimated according to the method of Porth et al. (2013). 100 mg of ground,
extract-free stem tissue was mixed with 3.5 ml buffer (60 ml of glacial acetic acid, 1.3 g NaOH/L) and
1.5 ml of 20% sodium chlorite, and then gently mixed at 50 °C for 16 hours. The reaction was
repeated again and the residual sample was transferred to crucible to determine holocellulose
gravimetrically. -cellulose content was then quantified by reacting 30 mg of holocellulose with
2.5ml of 17.5% NaOH for 30 min, followed by a second 8.75% NaOH reaction for an additional 30
min. Then -cellulose was finally determined gravimetrically.
Lignin monomer composition was estimated by thioacidolysis as previously described by Robinson
and Mansfield (2009). Statistical analysis was performed by one-way ANOVA followed by Tukey’s
post hoc test, using three biological replicates and two technical replicates.
Total RNA isolation and quantitative RT-PCR
The basal 0-11cm of 8-week-old inflorescence stems were frozen and homogenized. Total RNA
isolation and subsequent cDNA synthesis was completed as per Liu et al., (2014). For qRT-PCR
analysis of the basal stem segments, PCR amplification was performed using a CFX ConnectTM real-
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time system (Bio-Rad), using 40 quantitative PCR cycles that were run under the following
parameters: denaturation step, 95°C for 20 sec; annealing step, 55°C for 30 sec; elongation step,
72°C for 1 min. ACTIN2 was used as the reference housekeeping gene. All primers are listed in Table
S1 and all gene accession numbers are listed in Table S2. The calculation of differences in gene
expression was according to Bhargava et al. (2010). Three biological replicates consisting of three
technical replicates were used for each analysis.
RNA-seq analysis
Total RNA was extracted from wild type, knat3, knat7, and knat3 knat7 inflorescence stems (1-15cm
from the top) using TRIZOL reagent (Invitrogen, Life Technologies) following the manufacturer’s
protocol. In total, 12 samples were sent to UBC-Djavad Mowafaghian Centre for Brain Health
(DMCBH) Next Generation Sequencing Centre for library preparation and Ion ProtonTM
semiconductor-based transcriptome sequencing (4 genotypes by 3 biological replicates).
The sequencing quality of the RNA-seq reads was evaluated by FastQC software
(http://www.bioinformatics.babraham.ac.uk/projects/fastqc), and clean reads were mapped to the
A. thaliana TAIR 10 reference genome using STAR (https://github.com/alexdobin/STAR). FPKM
(fragments per kilobase of exon per million fragments mapped) was used to estimate the gene
transcript abundance. Cuffdiff (Trapnell et al., 2012) was used to calculate FPKM and identify
differentially expressed genes across genotypes. GO functional enrichment analysis was performed
using PANTHER (https://www.arabidopsis.org/tools/go_term_enrichment.jsp) with a significance
level of P < 0.05 (Mi et al., 2013). The metabolism overview of DEGs between mutants and wild type
was detected by MAPMAN software (http://mapman.gabipd.org/).
DATA STATEMENT
All relevant data can be found within the manuscript and its supporting materials.
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ACKNOWLEDGEMENTS
We would like to dedicate this work to Dr. Carl Douglas, a wonderful friend and mentor, who’s vision
and insight stimulated this work. We thank Dr. John L. Bowman (Monash University) for providing
seeds of knox2 mutants and 35S::KNAT3 overexpression lines. We thank the Arabidopsis Biological
Resource Center for seeds of the T-DNA insertion mutant. We thank Kyra Janot (University of British
Columbia) for aiding with biomechanical tests and Dr. Faride Unda for the chemical analysis support.
We thank Sean Shang for the help of mRNA-seq data analysis. This work was supported by the
NSERC (Natural Sciences and Engineering Research Council of Canada) CREATE Grant “Working on
Walls” and Dr. Carl Douglas’s NSERC Discovery Grant. Support for SW from the University of British
Columbia Faculty of Science following Dr. Carl Douglas’ death is gratefully acknowledged.
CONFLICT OF INTEREST
The authors have no conflicts of interest to declare.
AUTHOR CONTRIBUTION
SW conceived and performed the experiments, analysed the data, and wrote the article, as well
aided in the design of the research plans; MY and EG participated in some experiments; Carl Douglas
(CJD), and ALS, PM and SDM participated in conception of research plans, in the supervision of the
experiments, and writing of the article.
SUPPORTING INFORMATION
Additional supporting Information may be found in the online version of this article:
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Figure S1. Cross-sections of stem vascular bundles in wild-type and different combinations of knox2
mutants.
Figure S2. Plant morphology of wild-type, knat3 knat7, knat4 knat7, and knat5 knat7.
Figure S3. Expression patterns of KNAT3 and KNAT7 in the root and stem.
Figure S4. MAPMAN metabolism overview showing different gene expression levels in knat3 and
knat7 stems compared with wild type (WT).
Table S1. Oligonucleotides used in this study.
Table S2. Genbank accession numbers for genes used in this article.
File S1. RNA-seq data of using knat3, knat7 and knat3 knat7 stems compared to wild type.
File S2. Differentially expressed genes in knat3 knat7 double mutants versus wild type in metabolic
pathways identified by MAPMAN.
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FIGURE LEGENDS
Figure 1. Expression patterns of KNOX2 genes in Arabidopsis stems.
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Histochemical localization of ProKNAT::-GLUCURONIDASE (GUS) activity in 8 week-old inflorescence
stems of ProKNAT3::GUS (a, e, i), ProKNAT4::GUS (b, f, j), ProKNAT5::GUS (c, g, k), and ProKNAT7::GUS (d, h, l)
transgenic plants. Results shown are representative of more than 3 independent lines. (a-d) hand
cross-sections from young upper inflorescence stems, and GUS signals were found in the cortex and
vascular bundles. (e-h) hand cross-sections from the middle of inflorescence stems, showing GUS
expression in cortex, interfascicular fibers, and vascular bundles. (i-l) hand cross-sections from basal
inflorescence stems. The promoter activities at the basal stems were detected in cortex, phloem and
protoxylem, but not in the fiber cells. co, cortex; if, interfascicular fibers; p, phloem; x, xylem. px,
protoxylem; mx, metaxylem. Scale bars = 30μm.
Figure 2. Xylem vessel morphology of wild-type (WT) and KNOX2 mutant plants.
Cross-sections of basal stem vascular bundles stained with phloroglucinol from 8-week-old WT (a),
knat3 (b), knat4 (c), knat5 (d), knat7 (e), and knat3 knat7 (f) plants; Arrows indicate collapsed xylem
vessels. Scale bars = 30μm.
Figure 3. Plant morphology and stem biomechanical properties of wild type (WT), knat3, knat7
and knat3 knat7.
(a) Fifty-day-old knat3 knat7 plants had a pendant stem phenotype compared with WT, and knat3
and knat7 single mutants. Bar = 10cm. Tensile strength (b), tensile stiffness (c), and tests show that
the bases of fresh stems (0-60mm from the base) of knat3 knat7 had reduced tensile strength and
tensile stiffness compared to WT or single mutants. 3-point flexural tests measured the stress at
yield (d) and the modulus of elasticity (e) of both fresh and dried stems at 15mm from the base.
White bars, WT; light-grey bars, knat3; dark-grey bars, knat7; black bars, knat3 knat7. The error bars
represent means ± SD. Statistical differences among the samples are labeled with different letters
(n=5-10; P<0.05, one-way ANOVA followed by Tukey’s post hoc test).
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Figure 4. Interfascicular fibers of wild type (WT), knat3, knat7 and knat3 knat7 inflorescence stems.
Cross-sections from the bases of stems of 8-week-old WT (a), knat3 (b), knat7 (c), and knat3 knat7
(d) plants stained with phloroglucinol. (e) Quantification of fiber wall thickness in WT, single and
double mutants. knat7 had a significant increase in interfascicular fiber wall thickness and the
double mutants knat3 knat7 had a dramatic decrease in interfascicular fiber wall thickness.
Statistical differences among the samples are labeled with different letters (P<0.01, one-way ANOVA
followed by Turkey’s post hoc test). Error bars = 2 × SD. n= 30-50. Bars = 20μm.
Figure 5. Lignin composition of wild type (WT), knat3, knat7, and knat3 knat7 stem cell walls.
(a) Syringyl lignin to guaiacyl (S/G) lignin monomer ratio of dried stem bases (0-11cm from the base).
knat3 knat7 stem had significantly lower S/G lignin ratio. Error bars represent standard deviations;
Statistical differences among the samples are labelled with different letters (P<0.01, one-way
ANOVA followed by Turkey’s post hoc test). n = 3.
(b) Mäule stain produces red colour in syringyl lignin-rich cell walls. Cross-sections from the bases of
stems of 8-week-old WT, knat3, knat7, and knat3 knat7 plants. The double mutant knat3 knat7 had
less syringyl lignin deposition. Scale bars = 30 μm.
Figure 6. Complementation of knat3 knat7 phenotypes with ProKNAT3::KNAT3-GFP and
ProKNAT7::KNAT7-GFP.
(a) Xylem vessel morphology of wild type, knat3 knat7, and complemented lines. Cross-sections of
basal stem vascular bundles with Mäule staining from 8-week-old plants show that the irx
phenotype of knat3 knat7 was complemented. Scale bars = 30μm.
(b) Fifty-day-old plants show the pendent phenotype of knat3 knat7 was complemented with both
gene constructs. Bar = 10cm.
(c) and (d) showing the interfascicular fibers in wild type, knat3 knat7, and complementation lines.
The reduced wall thickness in knat3 knat7 was restored to an increased level relative to the wild
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