Arabidopsis overexpressing OgAPX exhibited superior capacity for scavenging
H2O2 and displayed the same flowering time under 22°C (Fig.12, 14) but accelerated
flowering under 30°C compared with wild type (Fig. 16,19). APX affecting the AsA
status was well-characterized in thermal response and desirable to detail address its
function in orchestration of AsA and temperature on flowering process. Transcriptional
profiling elucidated versatile hierarchies in response to AsA and temperature
coordinating flowering, including oxidoreductases, heat response proteins,
phytohormone response proteins, and numerous transcription factors. The temporal
expression pattern at 30°C and expression levels under different AsA level mutants
speculated the critical role of stress-related MYBs and AP2 participating in redox state
functioning on a mechanism prior to sugar and miRNAs affecting flowering process.
The activity of APX would be accelerated when plant undergoing elevated
temperature condition and brought about severe deprivation of AsA (Bonifacio et al.,
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2011). OgAPXOX transgenic plants displayed similar AsA redox ratio at 22°C but
decreased pattern at 30°C compared with wild type in LD photoperiod (Fig. 10). It
reveals that utilization of AsA by APX would be reinforced swiftly under LD-30°C and
brought about early flowering in transgenic plants compared with wild type. MYBs,
such as AtMYB11, AtMYB12 and AtMYB111were validated their function in response to
the fluctuate of redox ratio resulting from change of environment (Weisshaar et al.,
2007; Grotewold et al., 2010). The expression level of AtMYB70 was increased in wild
type under LD-30°C, and it would be enhanced in OgAPXOX (Fig. 25). Therefore, we
suggested the AtMYB70 was a critical recipient sensing the drastic deceased redox status
and function on flowering. Redox control in R2R3-MYB was through to alter Cys
residues to form an intra-molecular S-S bond to influence MYB domain structure.
AtMYB70 belongs to R2R3-MYB. Therefore, OgAPXOX displaying drastic decease of
redox status under elevated growth temperature would carry out significant alteration of
Cys residues for influencing the expression of responsible genes.
Furthermore, a validated MYB binding cis element locating on QQS promoter
region (Li et al., 2009), and it infers that QQS transcription is also regulated by direct
binding of MYB within QQS gene promoter.
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Moreover, earlier studies have shown that flowering induction might be caused
by a combination of sugar import and mobilization of various polysaccharides (Lejeune
et al., 1993; Sulpice et al., 2009). QQS functioning on starch biosynthesis (Li et al.,
2009). OgAPXOX displayed drastic decrease of expression level of QQS than wild type
under LD-22/30˚C (Fig. 25,26). It infers that starch content is presumable also less than
wild type. Noteworthily, QQS would be responsive to AsA state rather than elevated
growth temperature. It might indicate that AsA play a vital importance for
thermal-induced flowering and carbohydrate homeostasis.
Interestingly, the flowering process was regulated by miR156 level and
carbohydrate metabolism (Schmid and Srikanth, 2011). It is tempting to speculate that
miR156 and it target SQUAMOSA PROMOTER BINDING PROTEINLIKE (SPL) would
be response to the carbohydrate status of plant under endo- or exo- environmental
events. It suggests that QQS regulated by AsA state provides a prior regulatory
hierarchy in the cross-talk miRNA and carbohydrate on flowering process.
Moreover, phase transition in Arabidopsis would be promoted when plant
presented decreasing miR156 level and further trigger SPL3 expression level (Wu and
Poethig, 2006). SPL3 was direct upstream activator of LEAFY (LFY) (Yamaguchi et al.,
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2009).The expression level of SPL3 was significantly increased in OgAPXOX after 1
day under LD-30°C (Fig. 24). It suggests that the increasing SPL3 is a consequence of
decrease in miR156 level presumably. SPL protein promoted the miR172 transcription
to trigger downstream signal cascade for phase transition (Wu et al., 2009; Zhu and
Helliwell, 2011).
MiR172 acted as downstream of SPLs to repress six AP2-like transcription factor,
such as TARGET OF EAT (TOE) proteins (TOE1, TOE2, and TOE3), SMZ and its
paralog SCHNARCHZAPFEN (SNZ) (Mathieu et al., 2009). The expression level of
TOE1was significantly down-regulated in OgPMEOX but up-regulated in vtc1 (Fig. 27).
However, OgAPXOX displayed drastic decrease of expression level of TOE1 than wild
type after 5 day under LD-30°C (Fig. 23). It suggests that TOE1 would be regulated by
AsA in different genotype background.
SMZ, other target gene of miR172, would require FLM to repress FT. In Fig. 20,
the lower expression level of AtFLM was present in OgAPXOX transgenic plants
compared with wild type after 5 day under LD-30°C and figured out the role of AsA
participating in flowering coordinating with thermal-induced pathway.
In summarize, the genetic network of OgAPXOX provide a complex regulatory
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mechanism of flowering by altering the redox state under elevated growth temperature
(Fig. 30).
Noteworthily, the clock-associated gene PSEUDO-RESPONSE REGULATOR9
(APRR9) has been validated in activation of CO expression and antagonistic to LATE
ELONGATED HYPOCOTYL (LHY) during the daytime (Nakamichi et al., 2007). The
expression levels of APRR9 and HEME ACTIVATOR PROTEIN 2B (ATHAP2B) were
significantly down-regulated in OgPEMOX whereas LHY was up-regulated in vtc1 (Fig.
28). However, OgAPXOX showed lower AsA level under elevated growth temperature,
but the flowering positive regulator APRR9 and AtHAP2B showed decrease and steady
pattern, respectively (Fig. 24). It suggests that these clock genes were not all response to
AsA status and brought about the differential expression pattern under different
genotype backgrounds. This results also support the previous evidence declaring
elevated growth temperature would not require the photoperiod effector CO to act
upstream of the floral integrator FT (Balasubramanian et al., 2006).
Moreover, the AP2 transcription factors in OgAPXOX transgenic plants displayed
stinking decreased gene expression compared with wild type under LD-30°C day 5 (Fig.
23). It reveals that AP2 transcription factors were regulated by the homeostasis of ROS
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and AsA in Oncidium and Arabidopsis (Shaikhali et al., 2008; Wang et al., 2008b). In
previous studies showed that RAP2.4 type genes were down-regulated by light but
up-regulated by salt and drought stresses (Wang et al., 2008b). However, transgenic
plant overexpressing RAP2.4 would present early flowering compared with wild type
(Lin et al., 2008). Although these RAP2.4 type AP2 validated in response to oxidative
stress were not be monitored in OgAPXOX transgenic plant under different conditions,
the expression levels of different RAP2.4 genes displayed significant relevancy with
AsA status, including of At4g39780, At1g64380 and At2g22200 , and speculates their
probable function on thermal-induced flowering.
The shorter inflorescence was another significant phenotype in OgAPXOX
transgenic plants. Moreover, the starch is provided energy to sustain floral
development (Wang et al., 2008a). In Fig. 25, we speculate the higher expression level
of AtMYB70 in OgAPXOX would bring about the decreasing AtQQS and further
affected the starch biosynthesis. Therefore, the damage of inflorescence growth could
be presumably perceived to the work of APX on carbohydrate mobilization under
elevated growth temperature condition (Fig. 17). Accordingly, we suggest that
AtMYB70 is not only function on flowering but also regulate the inflorescence growth
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attributing to carbohydrate metabolism. Moreover, adg1-1/tpt-1 double mutant
displaying severe impairment in starch biosynthesis and triose phosphate / phosphate
translocator not only resulted in decline maximum photosynthetic electron transport
rate but also diminished chlorophyll contents compared with wild type (Hausler et al.,
2009). It also appears that the chlorosis phenotype in OgAPXOX transgenic plants can
be attributed to the total concentration of starch in OgAPXOX is less than control
plants presumably (Fig. 3).
In summarize, the genetic network of OgAPXOX provides a complex regulatory
mechanism of flowering by altering the redox state prior to miRNA and sugar-affected
flowering under elevated growth temperature.
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