THE ROLE OF CYTOKININ AND

ABSCISIC ACID IN RICE GRAIN-

FILLING

Term Paper Submitted by:

Ms. Lilethee Uy Malabug

HORT 232
March 30, 2009
I. INTRODUCTION

The growth and development of rice is affected by several factors. Among

these are the artificial substances such as pesticides, fertilizers and plant
growth regulators. Plant hormones (phytohormones) or plant growth
substances are generally endogenous to the plants. Grain filling is one of the
most important developmental processes in cereals, since the process
directly determines final grain weight and grain yield (Kato et al., 1992). Plant
hormones are considered as key regulators to seed development (Davies,
1987; Brenner and Cheikh, 1995)

Cytokinin (CK) are mainly produced in root meristems, young leaves,

young fruits, and seeds and translocated to the shoot organs via xylem. While
many plant parts are also known to have the ability to produce CK (Letham,
1994), the major place of production is considered to be root. Cytokinin (CK)
was first discovered as a plant hormone that promotes cell division (Miller et
al., 1955). It is now known to influence various aspects of plant growth and
development, including seed germination, apical dominance, leaf expansion,
reproductive development, and delay of senescence (Mok et al., 1995).

Different factors contribute to grain production in rice one of which the

delay in leaf senescence. ABA (abscisic acid), an important growth regulator
during plant growth and development, has been demonstrated to be involved
in many plant developmental processes, especially seed development
including maturation and germination, and plant response to environmental
factors (Lin et al., 2003).

II. GRAIN FILLING AND ITS REGULATION BY ENDOGENOUS

CYTOKININ (CK) AND ABSCISIC ACID (ABA) IN RICE

A. The Rice Grain Filling

A basic knowledge of the path of assimilate transport and storage, and

development of endosperm and embryo will help in improvement of yield and
grain quality in rice (Krishnan and Dayanandan, 2003). The process of grain
filling is actually the increase in cell number and cell filling in the endosperm
(Yang et al., 2002). Phytohormones, cytokinin, in particular is one of the many
factors affecting the development of rice grain. Below is a representation of
the development of a rice seed from pollination to seed maturation. The rate
and process of grain filling occurs at most usually at the first few days after
anthesis. Cytokinin usually functions at its best and found to have its
maximum level at the first few weeks of grain development while ABA would
be at around the middle week of grain development.

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 Figure 1. Size and shape of rice caryopsis at 2–3 day intervals during
development from anthesis (upper left) to maturity, 30 days after
fertilization (DAF) (lower right).

B. Levels of Endogenous CK in rice

The levels of endogenous CK reported in rice vary among organs and

developmental processes. There are reports that the highest concentrations
of CKs in rice are generally found in developing seeds (Yang et al., 2002). In
a study conducted by Yang et al. (2002), Zeatin (Z) and Zeatin riboside (ZR)
contents increased in the endosperms of spikelets during early endosperm
development. Like the endosperms, the levels of Z and ZR contents in the
roots were related to rate of endosperm cell division. The faster the rate of
endosperm cell division, the more Z and ZR in the roots during early
endosperm development. The rate of endosperm cell division was
significantly and positively correlated with the changes in Z and ZR contents
in roots and endosperm (Yang et al., 2002).

Morris et al. (1993) reported that zeatin (Z) and zeatin riboside (ZR) in
developing rice (Oryza sativa) and wheat (Triticum aestivum) grains showed
large transient increases following pollination, which coincided with the period
of seed setting and maximum endosperm cell division. This might be the
reason why CKs are said to positively affect grain development through.

Aside from its effect on rice grain-filling, CKs are known to be effective in
retarding senescence which indirectly affects grain production in rice. The
longer period that senescence occurs, the longer time for production and
allocation of food into the maturing grains. CKs might participate in the control
of meristem identity, in particular, to maintain the identity as the branch
meristem in panicle branching (Kyozuka, 2007). An increase in panicle
branching was observed in some of the varieties they have studied. The
positive effect of CK on panicle branching will increase the density and
number of spikelets hence increasing the overall yield of the crop.

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 C. Levels of Endogenous ABA in rice

In a study conducted by Kato et al. (1992) on two rice cultivars, the

content and concentration of endogenous ABA in developing grain increased
after heading, and attained a maximum value at about the middle of the
linearly increasing period of grain dry weight. The higher ABA content was
found with the cultivar which showed a larger grain size and a higher grain
filling. Kato et al. (1993) reported that ABA content in large-size grains was
higher than that in small-size grains during rice grain filling. Wang et al. (1998)
and Yang et al. (1999) suggested that the poor grain filling was associated
with low IAA and ABA contents in rice grains.

D. Regulation of Rice Grain-filling by Endogenous CK

CK activity like ABA in plants is controlled by a balance between several

factors, which include synthesis, degradation, inactivation, reactivation, and
other internal and external factors. CKs are supposed to be involved in long-
distance translocation through the selective transport system via xylem and
phloem, making the mechanism of regulation of CK activity more elaborate
(Kyozuka, 2007).

Kinetin can function in grain-filling indirectly through regulation of

senescence and the development of adventitious roots, thus a new supply of
CKs. The capacity of the roots for CK synthesis is an important determinant of
the ability of rice plants to maintain photosynthetic rate in leaves during
ripening particularly in high-yielding rice cultivars (Ookawa et al., 2003).

The delaying of senescence can potentially increase yield of the crop

through prolonging photosynthesis, thus increasing the amount of food
diverted to the sink during the ripening stage of the crop (Gan and Amasino,
1996).

Since the endosperm cell number in cereal crops is established during the
early phases of grain development, it is speculated that this parameter may
be regulated by CK rather than by the levels of available carbohydrates
during grain filling (Jones et al., 1992). Aside from the increase in cell number,
CK accumulation in the inflorescence meristem can explain the significantly
higher grain numbers (Ashikari et al., 2005).

Natural CKs such as trans-zeatin (tZ) and isopentenyladenine (iP) are N6-
substituted adenine derivatives that generally contain an isoprenoid side

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chain (Mok et al., 2001). CKX, is an enzyme known to preferentially and
irreversibly degrades nucleobase CKs by cleavage of the unsaturated N6-
isoprenoid side chains (Mok et al., 2001). This catabolic enzyme probably
plays the principal role in controlling CK levels in plant tissues (Armstrong,
1994; Werner et al., 2001; Werner, 2003).

Ashikari et al. (2005) showed that a QTL that increases grain productivity
in rice, Gn1a, is a gene for cytokinin oxidase/ dehydrogenase (OsCKX2), an
enzyme that degrades the phytohormone cytokinin. Grain number per panicle
is an important agronomic trait in rice (Kyozuka, 2007). Grain number1a
(Gn1a), a QTL locus that controls grain number in rice was isolated and
shown to encode OsCKX2, a CK degradation enzyme (Ashikari et al., 2005).
The negative correlation between OsCKX2 expression level and grain
number indicates a positive role of CKs on grain number (Ashikari et al.,
2005). The reduced expression of OsCKX2 causes cytokinin accumulation in
inflorescence meristems and increases the number of reproductive organs,
resulting in enhanced grain yield. In a study conducted by Ashikari et al.
(2005), the highest levels of OsCKX2 expression in inflorescence meristems
were found in Koshihikari. Transcript accumulation was less abundant in
Habataki and NIL-Gn1a and extremely low in 5150. These differences
indicated a correlation between OsCKX2 expression levels and grain number.
They also suggested that the phenotypic differences observed might have
been caused by differential transcription of OsCKX2. The expression of
OsCKX2 in inflorescence meristems might regulate the CK level to control
flower number (Ashikari, 2005). CK is known to be translocated acropetally
via the xylem and systemically via the phloem (Bernier et al., 1993). The high
levels of expression in these tissues suggest that OsCKX2 plays a role in
regulating CK levels in the vascular system of developing culms, where CK is
transported to the inflorescence meristems (Ashikari, 2005), thus functions in
inflorescence developement. In their study, OsCKX2, have been concluded
as a gene that increased grain number by ~21%. Gn1a encodes OsCKX2,
alleles of OsCKX2 encode functional enzymes, an enzyme that degrades
bioactive CK.

Aside from the regulation of grain filling through the effects of genes that
encodes enzymes degrading it, CKs could account for the difference in the
reduction in leaf level of Rubisco during senescence between rice
Cultivars(Ookawa et al., 2004). The rate of photosynthesis during senescence
is closely correlated with leaf levels of ribulose- 1,5-bisphosphate
carboxylase/oxygenase (Rubisco) (Makino et al., 1985). There is also a close
correlation between nitrogen content and Rubisco content during leaf
senescence (Makino et al., 1984). The larger amount of cytokinins
transported from roots to the shoot might cause the higher leaf level of
Rubisco and partitioning more nitrogen to leaves (Ookawa et al, 2004).
Ookawa et al. (2004) proposed that cytokinin regulates Rubisco content
directly by affecting Rubisco gene expression at the leaf level and indirectly

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nitrogen partitioning at the whole plant level. It can be assumed that CKs
might affect the nitrogen transport, assimilation and re-assimilation in source
and sink organs and therefore change the partitioning of nitrogen between
source and sink organs (Ookawa et al, 2004). The effect of cytokinin on both
the synthesis of Rubisco in leaves and the partitioning of nitrogen to leaves in
rice plants during ripening suggests that cytokinin causes the differences in
Rubisco reduction during senescence, thus resulting in differences in the
reduction of photosynthesis between two rice varieties, Akenohoshi and
Nipponbare (Ookawa et al., 2004). The capacity of the roots for cytokinin
synthesis is therefore an important determinant of the ability of rice plants to
maintain photosynthetic rate in leaves during ripening in high-yielding rice
cultivars (Ookawa et al., 2004).

In a study conducted by Yuan et al. (2008), OsMT2b (Oryza sativa

Metallothionein 2b), a gene that was characterized molecularly and found that
its expression was down-regulated by cytokinins. OsMT2b was preferentially
expressed in rice immature panicles, scutellum of germinating embryos and
primordium of lateral roots. Localization of isopentenyladenosine (iPA), a kind
of endogenous cytokinin, in roots and germinating embryos expanded to the
whole tissues, while in overexpressing plants, the iPA signals werevery faint
in the vascular tissues of roots and scutellum cells of germinating embryos
indicating a possible feedback regulation mechanism of OsMT2b to the level
of endogenous cytokinins which is involved in root development and seed
embryo germination of rice (Yuan et al., 2008).

E. Regulation of Rice Grain-filling by Interaction of Endogenous

CK and ABA with Changes in N and Soil Moisture Regimes

In a study conducted by Yang et al.

(2001), two different nitrogen level
(High N and Normal N) and two
different soil moisture regimes (Well-
Watered and Water-stress) were
tested on rice to evaluate their effects
on rice grain filling and the levels of
different hormones affecting grain
filling and seed development. The
differences in rice grain filling process
and rate in different treatments were
presented in the figure on the right
side. The grain filling process were
almost the same for all treatment. As
for the grain filling rate, it peaks at
different periods.

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 Results of an experiment conducted by Yang et al. (2001) showed that, Z
_ ZR contents in the grains transiently increased at early grain filling stage,
and reached a maximum at 9 to12 DPA at NN and 12 to15 DPA at HN, and
decreased thereafter. It was observed that during the first week of withholding
water (9–15 DPA), the difference in Z _ ZR contents was not significant
between WW and WS treatments. Under WW, the grains contained more Z _
ZR than under WS treatments only at the midand late-grain filling stages (18
DPA afterward). The Z _ZR contents were lower in HN grains than in NN
grains at early grain filling stage (3–15 DPA). At mid- and late-grain filling
periods, the difference was reversed and HN grains had more cytokinins than
NN ones.

Yang et al. (2001)

ABA content in the grains was low and slowly increased at early grain
filling stage, and reached a maximum at 15 to18 DPA under NN and 18 to 21
DPA under HN treatments (Fig. 4, C and D).

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Yang et al.(2001)

Water stress remarkably enhanced ABA accumulation in the grains at both

NN and HN. At early and mid-grain filling stages, the grain with NN contained
more ABA than those with HN treatments when the _soil was kept at the
same level. The increases and declines of ABA content in the grains
coincided with the grain filling rate. The time for ABA to reach the peak value
was simultaneous with the time of the maximal grain filling rate. ABA
accumulation in the grains was also associated with the partitioning of the
14C into the grains. The peak values of ABA in the grains were positively and
significantly correlated with the maximum grain filling rates and the
partitioning of the 14C into grains. Cytokinin (zeatin _ zeatin riboside) and
indole-3-acetic acid contents in the grains transiently increased at early filling
stage and WS treatments hastened their declines at the late grain filling stage
(Yang et al.,2001). Abscisic acid (ABA) in the grains was low at early grain
filling but WS remarkably enhanced its accumulation.

As a response to these variations in nutrient content and soil moisture

regimes, the Orysa sativa Response regulator (OsRR) genes will be induced
by cytokinin even in the absence of de novo protein synthesis and this
qualifies them to be primary cytokinin response genes (Jain et al., 2006). The
induction of OsRR6 in response to different environmental stimuli such as
nutrient and water stresses indicates its role in cross-talk between abiotic
stress and cytokinin signaling (Jain et al., 2006).

III. CONCLUSION

The different levels of endogenous CK and ABA are associated with rice
grain-filling. Results of most experiment showed that an increase in grain-

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filling corresponds to the increase in the level of endogenous CK and ABA
Endogenous CK and ABA are said to be regulated by different factors such as
genes controlling them and the different environmental stimuli or abiotic
factors. Further understanding of the CK and ABA, and their interaction with
different factors would be a prerequisite for a better understanding their
function during the different plant processes development.

IV. FUTURE PROSPECTS

QTL pyramiding to combine loci for grain number and plant height in the
same genetic background generated lines exhibiting both beneficial traits and
these results provide a strategy for tailormade crop improvement (Ashikari et
al., 2005). Further research and studies are also needed in interaction of the
different hormones and their various effects to plant development. More
studies on the involvement of genes regulating them are also needed.

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