1. Auxin and Cytokinin Cross-talk at the Level of Gene Expression in Arabidopsis thaliana
Steven Bench and Dennis Mathews
Department of Biochemistry, Molecular, and Cellular Biology
University of New Hampshire, Durham, NH 03824
Abstract Cytokinin Signaling WT arr10 arr1 arr1,12 Results: DR5:GFP Expression
 Auxin and cytokinin are plant phytohormones that play a  Cytokinin signaling in Arabidopsis consists of a two-  Using the synthetic auxin response element DR5, which is
significant role in organ formation and cell division in component system which involves a histidine kinase and a fused to the GFP gene promoter, (Ulmasov et al., 1997) we
plants. It is known that exposing a plant tissue cell response regulator. Cytokinin binds to the sensor domain of were able to see the instantaneous activity of auxin in the
MS
culture to auxin will promote root growth whereas the kinase receptor, then is autophosphorylated. Then, the plant root tips.
cytokinin will promote shoot growth. Less is known, kinase phosphorylates a histidine-containing  Figure 4 shows an extended DR5 expression in the WT and
however, about how these hormones interact with each phosphotransfer protein (Htp), which can enter the arr10 seedlings in the columella and epidermal cells. This
other at the molecular and cellular level. In order to nucleus. Next, the Htp phosphorylates an aspartate residue pattern is seen somewhat in the arr1 mutant, but not at all in
discover more about this relationship, we have studied on the response regulator (Hutchinson, et. al 2002). the arr1,12 double. Because the double mutant is more
plants that are compromised in their cytokinin signaling
Response Regulators BA insensitive to cytokinin, we can conclude that the extended
ability because of knockout mutations in certain
GFP expression is a direct result of cytokinin activity.
transcription factor genes known to be regulated by  In this study, we focused on type-B Arabidopsis Response
cytokinin. We have also used reporter constructs (GUS Regulators (ARRs), which can act as transcription factors  Additionally, the arr1 single mutant is more insensitive to
and GFP) to visualize the expression of genes known to once activated by the cytokinin-initiated phosphorylation cytokinin than the arr10 mutant, which lead us to conclude
be activated by auxin. With these research tools we have cascade. There are 23 identified type-B ARRs although here the ARR1 gene is more important in cytokinin signaling.
Figure 4 DR5:GFP expression in 5-day old seedlings. Plants
been able to study the effect of cytokinin signaling on the we consider only numbers 1, 10, and 12, which are thought were grown on either MS media or 1µM cytokinin (BA).
sensitivity of plants to auxin. to be the most important in cytokinin signaling. This
 The purpose of this study is to examine the interaction signaling pathway can be seen in Figure 1.
between cytokinin and auxin; in particular, the effects of
WT 1,12 1,10,12 Results: DR5:GUS Expression
decreased cytokinin signaling on auxin activity.
 The second reporter gene used was beta-gulcuronidase
MS 1µM BA
ARR Mutants (GUS). The GUS enzyme reacts with auxin to form a
MS permanent blue pigment, allowing us to see the total
 In order to observe the effect of cytokinin on genes
accumulation of auxin over the 5 days of growth.
known to be activated by auxin, we used a series of plant
lines containing knockout mutations in the alleles for  In Figure 5, it is clear that the GUS expression in the WT root
cytokinin signaling. T-DNA insertions were used in genes on cytokinin for 5 days is eliminated in both the arr1,12 and
to knock out their activity, which results in a significant the arr1,10,12 mutants. Interestingly, the level of GUS
decrease in the primary root. By decreasing the plant’s expression seems to increase in the arr1,12 mutant when
1 µM BA (2 hours)
exposed to cytokinin for 2 hours, but is reduced when grown
ability to complete the cytokinin signaling pathway, we
can observe the effects that result on auxin activity. on cytokinin for 5 days.
 We have utilized a number of different mutant plant lines  The wildtype seedlings show that the DR5 promoter is
that have varying degrees of cytokinin signaling upregulated by cytokinin, which was unexpected because
inhibition. For example, the arr1,10,12 mutant contains Figure 2 DR5:GFP expression in 5-day old wildtype plants on DR5 was designed synthetically to respond to auxin.
mutations in the those three alleles, and is therefore MS and cytokinin (BA). Upon addition of BA, the seedlings 1 µM BA (5 days)
show a significant increase in auxin activity primarily in the
more insensitive to cytokinin than the arr1 mutant,
vascular tissue and the outer epidermal cells.
which only has a mutation in the ARR1 allele.
Figure 5 DR5:GUS expression in 5-day old seedlings. Plants
exposed to BA for 2 hours were initially grown on MS media.
Conclusions
 Decrease in cytokinin signaling results in a substantial redistribution in auxin activity, most notably in
the vascular tissue and epidermal regions.
 Cytokinin is important in regulating the activity of auxin in the root meristems.
 The DR5 response element, which was designed to be activated by auxin, is also activated by cytokinin.
References
 Hutchinson, C.E., Keiber, JJ. (2002). Cytokinin Signaling in Arabidopsis. The Plant Cell, 14, 547-559.
 Jaillais, Y. (2012). Arabidopsis Root Tip Anatomy [online] Available: http://www.ens-lyon.fr/RDP/SiCE/Resources.html
 Ulmasov, T., Murfett, J., Hagen, G., and Guilfoyle, T. (1997). Aux/IAA Proteins Repress Expression of Reporter Genes Containing
Figure 3 Arabidopsis root tip anatomy (Yvon Jaillais, 2012) Natural and Highly Active Synthetic Auxin Response Elements . The Plant Cell, 9, 1963-1971.
Figure 1 Cytokinin signaling in Arabidopsis