Changes of pip mutants in response to variable environmental scenarios

2.3 Involvement of PIP2;1 , PIP2;2 and PIP2;4 in responses to variable environmental

1, 1.5% 3, 4.5% 1, 1.5% 3, 4.5% 4, 6% 5, 7.5% 2, 3% 7, 10.4% 4, 6.0% 4, 6.0% 9, 13.4% 24, 35.8%

Major carbonhydrates metabolism Cell wall Lipid metabolism Secondary metabolism Signalling Transport Development

Miscellaneous enzyme families Biotic and abiotic stress RNA.Transcription

Protein modification and degradation Not assigned

1, 5.6% 1, 5.6% 1, 5.6% 1, 5.6% 1, 5.6% 2, 11.1% 11, 61.1%

Major carbonhydrates metabolism Cell wall

Lipid metabolism Secondary metabolism RNA.Transcription

Protein modification and degradation Not assigned

Changes of pip mutants in response to variable environmental scenarios

2.3 Involvement of PIP2;1 , PIP2;2 and PIP2;4 in responses to variable environmental

2.3.3 Changes of pip mutants in response to variable environmental scenarios

scenarios

2.3.3.1 Transcriptome profiling of pip mutants under well-watered

condition

To test the transcriptional changes in rosettes induced by loss of the major

PIPs and

additional root specific PIP expression under normal growth condition, Agilent microarrays

were employed for transcriptome analysis in pip2;1 pip2;2 and pip2;1 pip2;2 pip2;4 grown on

well-watered soil for four weeks (See 4.2.2.1). Transcripts having │Log

FC│≥ 1 and adj.P ≤

0.05 in pip mutants as compared to the wild type were selected as differentially expressed

genes and used for further analysis (See 4.2.4.1).

In total, 65 genes were differentially regulated in pip2;1 pip2;2. Among these genes, 17 were

induced and 48 were repressed. In pip2;1 pip2;2 pip2;4, only 18 genes were differentially

expressed including six upregulated genes and 12 downregulated genes (Figure 26A; Table 6).

Two upregulated genes and 11 downregulated genes were overlapping between the two

mutants. Consequently, 15 induced genes and 37 repressed genes were unique to pip2;1

pip2;2, but only four upregulated and one downregulated genes were unique to pip2;1

pip2;2 pip2;4 (Figure 26B; Table 6). These data indicate that greater transcriptional changes

as a response to the loss of PIP2;1 and PIP2;2 in the presence of PIP2;4.

To better understand the functional classification of the differentially expressed genes in pip

mutants, gene ontology (GO) analysis was employed using MapMan 3.5.1R2. The

differentially regulated genes were involved in major carbohydrates metabolism, cell wall,

lipid metabolism,

secondary metabolism,

RNA transcripts,

protein modification and

degradation in both pip mutants. In addition, the differential regulations in signalling,

53

responses were specifically displayed in

pip2;1 pip2;2. Moreover, a large number of

functionally uncharacterized genes were detected in both pip mutants(Figure 27; Table 6).

Among the differentially expressed genes of both pip2;1 pip2;2 and pip2;1 pip2;2 pip2;4

under well-watered condition, QUA-QUINE STARCH (QQS), which is a negative regulator of

starch accumulation, and DESULFO-GLUCOSINOLATE SULFOTRANSFERASE 18 (SOT18), which

is involved in the final step of glucosinolate core structure biosynthesis, were enhanced.

Among the transcripts that were specifically decreased in pip2;1 pip2;2, two genes encoding

cell wall surface modification, AGP5 and

PME17, as well as the transporter

CATION

EXCHANGER 3 (CAX3) was detected. In addition, the protein kinase

CYSTEINE-RICH

RECEPTOR-LIKE PROTEIN KINASE 7 (CRK7) was repressed. For specifically regulated genes in

pip2;1 pip2;2 pip2;4, no clear cellular function could be assigned (Table 6).

To extend the functional interpretation of the unknown genes, co-expressed gene networks

of identified differentially expressed genes in pip mutants were retrieved from ATTED-II

(http://atted.jp) (Figure 28; Table 6). Interestingly, the function unknown genes AT2G44240,

AT3G13950, AT4G10500 as well as AT1G26380 were strongly co-expressed with AGP5, CAX3,

two protein kinases CRK7 and RLP7 as well as three stress responding genes SAG13, PR1 and

YLS9 (Figure 28A). In addition, the function unknown gene

AT1G30700 was positively

co-expressed with PME17, CHX17 and defense responding gene KUNITZ trypsin inhibitor 1

(KTI1) (Figure 28B). The extended genes included in the co-expression network also

displayed a slight reduction in our data (Table 7). The coexpression networks suggest that

these functionally unknown genes may be involved in cell wall modification, ion homeostasis

or defense responses. In contrast to pip2;1 pip2;2, the transcripts altered in pip2;1 pip2;2

pip2;4

were not part of specific co-expression networks which could give additional

information on their functional implication.

Taken together, the transcriptome data showed that loss of the functional PIP2;1 and PIP2;2

induced the alterations associated to cell wall modification,

cell membrane compartments,

54

The alterations at the transcriptional levels were more pronounced in pip2;1 pip2;2 than in

pip2;1 pip2;2 pip2;4.

Figure 26. Global differentially expressed genes in pip mutants as compared to the wild type under

well-watered condition. (A) Number of differentially expressed genes in pip2;1 pip2;2 (DM) and

pip2;1 pip2;2 pip2;4 (TM). Red and green marks represent upregulated and downregulated genes,

respectively. (B) Venn diagrams showing the differentially expressed genes unique or common to

pip2;1 pip2;2 and pip2;1 pip2;2 pip2;4. Only transcripts with steady-state fold change │Log

FC│≥ 1

and adj.P ≤ 0.05 are included.

DM

17

48

65 genes

TM

6