Supplemental description
Integration of anatomical gene expression patterns in the HSP70 phylogenetic tree
To determine the functions of rice HSP70 family members, we integrated anatomical
expression patterns for 11 tissues/organs comprising 209 hybridizations (NCBI GEO series
GSE21398). In addition to the five HSP70 genes that lacked probes on the Agilent44K array,
this integration included anatomical expression profiles for 27 other HSP70 genes (Fig. S1).
Those Agilent44K data were represented by log2 normalized intensity values (Table S2).
Anatomical meta-profiling data based on 983 rice Affymetrix microarrays are shown in
Figure S2.
Cluster I
The phylogenetic tree indicated close links among Os05g38530, Os01g62290, and
Os11g47760. The first two had similar expression patterns in most analyzed samples,
indicating possible functional redundancy in a developmental or anatomical role (Fig. S1).
Their expression was also down-regulated under darkness (Fig. S1). The expression level of
Os01g62290 was much higher than Os05g38530, suggesting that the former is dominant.
Whereas Os05g38530 was preferentially expressed during early ovary development,
Os01g62290 showed greater expression in all other organs and tissues examined here (Fig.
S1). Constitutive expression and high transcript levels were noted with Os11g47760 (Fig. S1).
Neither Os12g38180 nor Os03g16920 was clustered with other members in this group; the
latter was more highly expressed in the stems, anthers, roots, and ovaries at 7 days after
pollination (DAP), as well as in maturing embryos and maturing endosperm, while the former
was constitutively expressed, usually at a moderate level, but with stronger expression being
observed in maturing embryos. Therefore, based on these patterns, we expect that
Os03g16920 has a significant role in maturing embryos and the endosperm (Fig. S1).
Os03g60620 and Os03g16860 were closely linked in the phylogenetic tree; the former
showed a high level of constitutive expression in most organs, with one exception: expression
was repressed in mature roots at 76 days after transplanting. Expression by Os03g16860 was
also high and constitutive, except for leaf tissues collected during stages when the panicles
(inflorescence) and pistils were developing (Fig. S1). Os03g16880 had very low expression
in most samples, making it difficult to predict any anatomical function. Tandemly duplicated
Os11g08460 and Os11g08440 also were very closely linked in the phylogenetic tree, with
dominant co-expression being observed in mature anthers. While Os11g08460 showed
preferential expression only in mature anthers, Os11g08440 was significantly expressed in
young panicles, early developing anthers, ovaries, and the endosperm (Fig. S1). This
suggested that the two genes have redundancy during mature anther formation, but that only
Os11g08440 has unique functions in the developing ovary and endosperm. Because the
cytosolic OsctHSP70s, Os01g49430, Os11g08445, and Os11g08470 were not available on
Agilent44K, we could not obtain their data.
In summary, our anatomical meta-profiling data for this cluster indicated housekeeping
functions by the constitutively expressed Os11g47760 and Os03g60620, as well as roles in
organ/tissue development for Os05g38530 (early ovary), Os11g08440 and Os11g08460
(anther), and Os03g16920 (maturing embryo and endosperm).
Cluster II
In this cluster, the greatest constitutive expression was found with Os02g02410
(BIP1/OsBIP3), suggesting a dominant anatomical role (Fig. S1). Os03g50250, Os05g30480,
Os08g09770, and Os05g35400 were preferentially expressed in anthers at the vacuolated
pollen stage (Fig. S1), implying strong functional redundancy in pollen development. Among
them, transcript levels for Os05g30480 were approximately 2- to 4-fold higher in the ovaries,
embryos, and endosperm when compared with the other three, indicating that it might have a
unique and significant role in organ development. Because Os01g33360 was not included on
the Agilent44K, we could not obtain any anatomical meta-profiling data.
Cluster III
Os05g23740 and Os12g14070, which encode chloroplast-type HSP70 (OscpHSP70) protein,
were closely linked in the phylogenetic tree and had similar expression patterns in most
samples. An exception was seen with Os12g14070, which was more highly expressed in the
endosperm (Fig. S1). In most samples, expression by Os05g23740 was greater than by
Os12g14070, except during the late stages of ovary and endosperm development. This
suggested that the former has a dominant function in vegetative tissues while the latter has a
more prominent role in more mature ovaries and embryos. A mutation of Arabidopsis
cpHsc70-1 and cpHsc70-2, encoding chloroplast HSP70 protein, shows defective phenotypes
in plant development and thermo-tolerance in germinating seeds (Su and Li 2008), implying
that their roles are similar to that of their rice homologs.
Close links on the tree and similar expression patterns were found between
Os02g53420 and Os03g02260, which encode the mitochondria HSP70 (OsmtHSP70) protein.
However, Os02g53420 was the more dominant. Although over-expression of OsmtHSP70
(Os02g53420) suppresses PCD death in rice (Vitte et al. 2004), a loss-of function study is
needed to clarify its functional roles. Because Os09g31486 is not part of Agilent44K, we did
acquire its anatomical meta-profiling data using affymetrix microarrays and identify the
similar expression pattern with that of Os03g02260 (Fig. S2), suggesting its functional
redundancy with Os03g02260.
Cluster IV
Three proteins belong to this cluster: Os12g05760, Os05g51360, and Os06g10990. All were
expected to be localized in the cytoplasm and ER. Of these, Os05g51360 had very low
expression overall. Os12g05760 and Os06g10990 showed similarly high expression in
anthers at the young microspore stage (Fig. S1). Compared with other cluster members,
expression of Os06g10990 was higher in the ovary and pistil, thus allowing us to conclude
that this gene has the predominant role.
Cluster V
As the single member in this cluster, Os03g11910 was preferentially expressed in mature
anthers, ovaries at 1 DAP, and maturing embryos and endosperm (Fig. S1). This pattern was
very similar to that of Os03g16920 in Cluster I. It is possible that these two genes have
functional redundancy because both encode a cytoplasmic HSP70 protein.
Cluster VI
The four nuclear HSP70 proteins (OsncHSP70) in this cluster are Os02g48110, Os06g46600,
Os01g08560, and Os05g08840. In the phylogenetic tree, Os02g48110 was positioned slightly
distant from the others, and was highly expressed in early ovaries, maturing embryos, and
endosperm. Of the other more closely linked genes, Os05g08840 showed constitutively high
expression in most analyzed samples, while the others had very low expression (Fig. S1).
Evaluating any functional redundancy among the four OsncHSP70 genes will necessitate
related gene-indexed mutants or transgenic approaches.
Evaluation of functional redundancy among rice HSP70 family genes
The multiple HSP70 proteins located within each sub-cellular organelle indicated that those
in the same clade were more likely to have functional redundancy. Although this can make it
more difficult to identify their roles via loss-of-function studies, mutations in genes with
predicted functional redundancy often reveal visible phenotypes. For example, we previously
identified defective phenotypes caused by the mutation of a member of the rice serine
hydroxymethyltransferase (SHMT) family, indicating that this member has a predominant
role in that family (Jung et al. 2008a; Jung et al. 2008b). Therefore a better strategy for
predicting a dominant member within a gene (sub)family would be to integrate transcriptome
data into its phylogenetic tree. No phenotypes have yet been identified that are caused by the
mutation of any single rice HSP70 gene except for BIP3/OsBip3 (Os02g02410), whose
suppression leads to defects in the endosperm (Wakasa et al. 2011). Results from gain-offunction studies have demonstrated that BIP3/OsBip3 (Os02g02410) encodes the HSP70
protein in the ER and cytosol, thereby modulating XA21-mediated innate immunity, while
the mitochondrial HSP70 gene (OsmtHSP70, Os02g53420) regulates heat- and H2O2-induced
PCD in rice protoplasts (Park et al. 2010), (Qi et al. 2011).
Because functions are not yet known for the remaining genes, we integrated the
Arabidopsis best-hit genes next to the GO terms in the cellular component category in order
to determine the functions of the rice HSP70 genes. Of those Arabidopsis genes, chloroplast
HSP70 (cpHSP70-1, At4g24280) has a role in protein importation (Su and Li 2010) while
cytoplasmic HSP70 (HSP70B, At1g16030) modulates thermo-sensitivity in response to heat
or cold. Overexpression of HSC70-1 (At1g16030) inhibits plant resistance to virulent and
avirulent pathogens, even if a loss of function by individual cytosolic HSC70 genes reveals
no defense phenotype (Jungkunz et al. 2011). Through comparisons with those Arabidopsis
best hits, we might predict that Os03g16920 plays a role similar to that of At1g16030 in
controlling thermo-sensitivity, and we might expect Os12g14070 to function like At4g24280
in importing chloroplast protein. However, sequence similarity does not always determine
functional similarity across species (Chikina and Troyanskaya 2011). Instead, exclusive use
of genome-wide transcriptome data provides more accurate and unbiased arrangements
(Chikina and Troyanskaya 2011). We believe that, by integrating the expression profiling
data of the entire HSP70 family within the phylogenetic context (phylogenomic analysis), it
is possible to predict more precise functions for an individual member.
Advantage of phylogenomics analysis and limitation of functional genomics analysis due to
functional redundancy
Our anatomical meta-profiling data for cytosolic HSP70 family in cluster I effectively
estimated different functions such as abiotic stress responses (Os05g38530, Os01g62290, and
Os03g16920), housekeeping functions (Os11g47760 and Os03g60620), early ovary
development (Os05g38530), anther (Os11g08440 and Os11g08460), and maturing embryo
and endosperm (Os03g16920). Until now, any of these were not functionally identified,
suggesting the possible functional redundancy among gene family members in this cluster. In
real, we did not observe the phenotypic defects in a T-DNA insertion line of an OsctHSP70-1.
The stimulated expression of Os01g62290 in the mutant was sufficient to compensate the
defect of Os05g38530, while expression by Os11g47760 was not affected by the
Os05g38530 mutation, indicating that its functional redundancy was restricted to
Os01g62290 but not expanded to Os11g47760.
On the other hands, the greatest constitutive expression of Os02g02410 (BIP1/OsBIP3)
in cluster II suggests its dominant role (Fig. S1). Gene knock-down (KD) analysis of
BIP1/OsBIP3 in the XA21 background has shown that the resistance response to Xoo is not
affected (Park et al. 2010), whereas the same analysis of BIP1/OsBIP3 under the control of
an endosperm-preferential promoter can alter seed phenotypes, including the intracellular
structure of endosperm cells, content of seed storage proteins, starch accumulation, and grain
weight, thus demonstrating a dominant role for BIP1/OsBIP3 in seed development (Wakasa
et al. 2011). Moreover, expression of other paralogs (Os03g50250, Os08g09770, and
Os05g35400) is up-regulated in a BIP1/OsBIP3 KD mutant, indicating that the robustness
feature that compensates for this gene defect stimulates the expression of other paralogs
(Wakasa et al. 2011). Even though we did not assay Os05g30480 transcripts in the
BIP1/OsBIP3 KD, we predict that expression of that gene was stimulated in a manner similar
to that of the other paralogs. Because expression in the endosperm was greater for
Os05g30480 than for the others, its compensating effect would be more significant. However,
that prediction requires further testing. Regardless, previous results suggested that this
compensatory effect might be temporary, because the BIP1/OsBIP3 KD line eventually
manifested defective phenotypes during seed development (Wakasa et al. 2011).