Transcriptional hallmark responses to P limitation conserved in
switchgrass
Previous research with other plant species identified conserved
transcriptional responses to P-stress (e.g. Misson et al., 2005; Bari et
al., 2006; Morcuende et al., 2007; Pant et al., 2009; Plaxton & Tran,
2011; Secco et al., 2013). Switchgrass exhibited many of the same
transcriptional responses to P limitation (Table S3 ). These
responses included induction of gene transcripts for (i) uptake and
transport of Pi and other inorganic ions,e.g. Pht1 , PHO1 ,KUP , NRT1 ; (ii) Pi salvage systems,e.g. PAPs ,
ribonucleases, GDPDs , phospholipid degradation genes; (iii)
alternative metabolic pathways that lower P requirements, e.g. inorganic
pyrophosphatases); (iv) sulfo- and glycolipid synthesis, e.g.MGD s, DGD s, SQD ; (v) redirection of carbon
metabolism; (vi) phytohormone synthesis/response pathways; (vii)
disease/pathogen stress–responses, e.g. Cysteine-rich RLK, CAPs;
and(viii) repression of gene transcripts associated with the
photosynthetic machinery and photosynthesis (TableS3 ).
Surprisingly, few DEGs encoding SPX-domain proteins and no microRNA399s
were among the annotated switchgrass transcripts, possibly reflecting
incomplete annotation of switchgrass genes. In fact, unannotated DEGs
represented between one-third and two-thirds of all P-responsive gene
transcripts, depending on treatment and cut-off values used
(Figure S1 ). Therefore, we investigated the many unannotated
DEGs through manual homology searches against transcripts/proteins from
other plant species. To this end, we chose strongly responsive
(>5-fold change) DEGs in shoots and roots of
moderately-stressed plants grown with 60 µM Pi supply, which likely
elicited many specific P-stress responses without triggering many
non-specific responses related to severe impairment of growth and
development (cf. Figure 5b; Figure S1; Table S4 ).
Among the 380 DEGs with at least 5-fold change in transcript abundance
in shoots, 234 were up-regulated and 146 down-regulated. Of the
up-regulated DEGs, 109 (47%) were annotated, and many of these were
homologous to P-responsive DEGs in other plant species (B.
distachyon , M. truncatula , O. sativa and A.
thaliana ). Some of these genes have established functions during
P-limitation, such as phosphate transporters or purple acid phosphatases
(Table S4 ), but many encode unknown or uncharacterized
proteins, or proteins with functions not previously linked to
P-limitation, such as three DEGs encoding Sua5/YciO/YrdC/YwlC proteins
required for tRNA modification (Yacoubi et al., 2009). Moreover, 127 up-
or down-regulated DEGs with gene identifier, showed no homology to
proteins or RNAs in other species. These observations point to unknown,
possibly switchgrass-specific transcriptional P-starvation responses.
For the 125 up-regulated, unannotated DEGs, we performed dedicated,
manual BLAST searches against genes from other species (clade
Viridiplantae). This revealed that 84 of these DEGs do have significant
homology to known proteins, many of which are encoded by P- responsive
genes. This includes six additional SPX-domain proteins, four
glycerophosphodiester phosphodiesterases, three purple acid
phosphatases, two PHO1-like phosphate transporters, two
monogalactosyldiacylglycerol synthases (MGD , involved in
glycolipid synthesis), two sulfoquinovosyl transferases (SQD ,
sulfolipid synthesis), another phosphoenolpyruvate carboxykinase, two
glucose-1-phosphate adenylyltransferases small subunits and an
ADP-glucose pyrophosphorylase required for starch synthesis
(Table S4 ). For 31 (25%) of the 125 up-regulated,
unannotated DEGs no protein homologs were found. It is possible that
some of these 31 DEGs represent P-status responsive microRNA primary
transcripts or other long non-coding RNAs (e.g. IPS1, TAS4;Franco-Zorrilla et al., 2007; Hsieh et al., 2009) that play important
roles during P-limitation, but can only be found with targeted searches
(see below), while other DEGs may represent novel aspects of
P-starvation responses in plants.
We also scrutinized strongly-responsive DEGs (> 5-fold
change) in roots of plants grown with 60 µM Pi. Among the up-regulated
annotated DEGs in roots were many related to well-known transcriptional
responses to P-stress, e.g. membrane lipid remodeling, phosphate uptake
and salvaging. An interesting observation was the upregulation of three
ent-copalyl diphosphate synthases, an ent-kaurenoic acid oxidase-like
protein and an ent-kaurenoic acid hydroxylase, all involved in
gibberellin biosynthesis (Table S4 ).