“HEAT STRESS DURING PLANT REPRODUCTION”

STUDENT IDENTIFICATION NUMBER: 4257838

SEX, FLOWERS AND BIOTECHNOLOGY

Dr. Zoe Wilson

Word count: 1494

1. Introduction

During the last decades, there was a significant increase in the level of greenhouse gases in the
atmosphere that, combined with the fast land transformation, have accelerated the global warning
process. Previously, some studies have reported the direct relation between the high temperature
stress in plants and the failure in their reproduction cycle, also the reduction of yields of crops of
interest have been mentioned (Hedhly et al., 2009).

Although, plants can evolve when they are exposed to heat stress (HS) environments, it is essential to
have a better understanding of the molecular and cellular mechanisms behind this response.
Breeders and molecular engineers have used the generated information to design plants that
become tolerant to HS, maintaining normal yields (Hedhly et al., 2009; Sage et al., 2015).

2. General considerations

HS can produce several damages in the plant including changes in the membrane fluidity,
destabilization of proteins, cytoskeleton and ribonucleic acid structures, inefficacy of enzyme
reactions highly susceptible to temperature fluctuations. Also, the accumulation of reactive oxygen
species (ROS) is induced which in high concentrations leads to the cellular death. Moreover, the level
of injury depends of the intensity and duration of HS (Barnabás et al., 2008; Zinn et al., 2010).

There are several mechanisms which are adopted by plants to cope with HS. They produce
dehydrated pollen grains and embryos which can stay in dormant state; also, changes in the
hormone levels have been observed, for example signaling pathways of salicylic acid, abscisic acid
and ethylene are activated during the stress (Zinn et al., 2010).

Finally, the activation of HS transcription factors (HSF) is important because they are key players for
triggering the expression of different genes, such as the ones that encode for heat shock proteins
(HSP) that function as chaperones stabilizing proteins to avoid the denaturation (Zinn et al., 2010).

3. Anther and pollen development in normal conditions

A common anther is composed by four lobes, each containing five cell layers. The central cell layer is
the place where the microspore mother cell develops. It will generate uninucleate microspores
through meiosis. These, after two consecutive mitosis divisions, give rise to mature pollen grains.
When the pollen wall is completely developed the tapetum and the middle layer undergo
programmed cell death, while the endothecium layer plays crucial role in the anther dehiscence
process to release the pollen (Fig. 1) (Sage et al., 2015).

Figure 1 Normal anther development and pollen release; M pollen mother cells; T tapetum; Mi Middle layer; E
endothecium; Ep epidermis; L lobe; S stomiun; * uninucleate, **binucleate and *** trinucleate microspores; Bars 10 um
(Sage et al., 2015)

4. Impact of HS over uninucleate spore development

Several articles have mentioned that uninucleate development stage in male reproductive cycle is
the most sensitive process in high temperature conditions (Chen et al., 2015; Sage et al., 2015).
Abortion has been reported (>70%) in this stage using microscopy in many species including wheat,
barley, sorghum, rice, tomato and cotton under temperatures ≥30°C (Fig. 2). Tapetum layer function
is critical to normal development of mature pollen grains because it provides carbohydrates to
uninucleate microspores; therefore, it is assumed that failures in the carbohydrate metabolism
decrease the starch levels in pollen grains and HS could be related to time accuracy of tapetum
programmed cell death (Bita and Gerats, 2013; Sage et al., 2015). Pressman et al. (2002) reported
that changes in pollen development in tomato due to variations in the carbohydrate concentration
were induced by HS.
Figure 2 A to F) Abortion of uninucleate microspores in different species exposed to 36°C; G to H) non-abortion of pollen of
three species at 26°C; *nucleus; Bars 5 um (Sage et al., 2015)

Transcriptomic analysis under HS has been performed in tomato (heat tolerant and sensitive) and
cotton to identify the genes that are up or down-regulated during HS. Surprisingly, the tolerant
tomato variety showed over-expression in some heat shock factors and heat shock proteins at
ambient temperature compared with the susceptible variety, concluding that those genes could be
responsible to the thermotolerance effect. On the other hand, in the sensitive cotton line, down-
regulation of genes encoding for DNA methylation and histone demethylation was observed,
concluding that different epigenetic modification occurs in the heat tolerant line (Sage et al., 2015).

Finally, rice anthers were exposed to ≥36°C/29°C (day/night) HS, resulting in the complete pollen
abortion; no changes were observed in the tapetum, starch level or invertase activity, although,
intensive observations showed the accumulation of H2O2 (ROS species) in the nucleus and wall of
uninucleate microspores which subsequently became necrotic leading to cell death. Also, the
presence of H2O2 in high levels could interfere with the redox balance prior to gene expression of
important genes such as those that work in the metabolism of lipids affecting the pollen wall
biosynthesis (Sage et al., 2015).
5. Meiosis alterations during HS

Several studies have reported negative consequences during meiosis due to HS in many crops, such
as tomato, barley, wheat. In barley, the detention of the cell division during meiosis have been
related with failures in the auxin biosynthesis and down-regulation of genes responsible of synthesis
of DNA polymerase, histones, 60S ribosomal proteins, helicases (Sage et al., 2015).

Moreover, it has been mentioned that HS produces early tapetum degradation (Zinn et al., 2010).
Another consequence of high temperature in this stage is the ROS-induced autophagic program cell
death that disintegrates tetrads producing no pollen grains in the anther lobe, such as in barley (Fig.
3) (Sakata et al., 2000).

Figure 3 Barley anther with no pollen grains; Bar 500 um; (Modified from Sakata et al., 2000)

6. Mature pollen and anther dehiscence during HS

After a process of HS with temperatures >30°C in the day and >29°C in the night, it is common that
developed anthers contain mature but not viable pollen, mainly due to the presence of ROS species
which can modify phospholipids and the pollen membrane structure. On the other hand, with
temperatures above 30°C, Brachypodium anther opening can be interrupted and in the worst case it
never opens (Fig. 4) (Harsant et al., 2013). It has been suggested that temperature could influence in
the endothecium layer, main responsible of the anther dehiscence and pollen release; also, it seems
that the process is delayed due to the lack of swelled pollen. Lastly, HS could stop the anther
dehiscence avoiding the lysis of the cell walls that are separating the adjacent anther locules trapping
the pollen. High quantities of H2O2 may act promoting cell wall rigidity (Sage et al., 2015; Wilson et
al., 2011).

Figure 4 Brachipodium distachion anthers; Bar 500 um; A dehiscence; B indehiscence; Bars 20 um; (Modified from Harsant
et al., 2013)

7. Overcoming HS: practical approaches

7.1 Conventional breeding

An inconvenient using this approach is the difficulty to control the environmental conditions in the
field; however, now there are control glasshouses available especially for HS screening. Another issue
is that different species show variable tolerant temperature rates. The fundamental basis of this
approach is to grow breeding lines under HS conditions and select individual lines with superior grain
yield. Many screening and selection methods have been developed including: heat tolerant index,
pollen and seed viability (Wahid et al., 2007).

7.2 Molecular approaches

There are two important techniques which have been used to obtain tolerant plants against HS:
marker assisted selection (MAS) and genetic transformation (GT). However, before applying MAS, it
is necessary to identify genetic markers that are ligated with QTLs, genes that are responsible to
provide HS tolerance, for example in Arabidopsis thaliana four QTL providing HS tolerance were
found (Mach, 2015).

GT and transcriptomic analysis have helped to understand the genetic and biochemical bases of HS
behavior. Many enzymes, genes and compounds related with HS have been identified; however, the
availability of those genes to control expression has led to the production of HS tolerant varieties in
some crops. Recently, studies to transform plants with genes encoding for enzymes that detoxify
ROS, such as SOD have been performed. Other examples are: transgenic tobacco carrying the Dnak1
gene from the cyanobacterium Aphanothece halophytica, express HS resistance. Plants transformed
with the BADH gene, over produce glycinebetaine, they have been considered as a promising method
to overcome HS. Finally, transformation with rubisco activase gene and heat shock factor genes are
being studied to generate HS resistance (Wahid et al., 2007).

Conclusion

It is estimated that per each °C of rise in the global temperature through the growing season, the
grain yields of the major crops including rice and maize will decrease from 5 to 17%. To guarantee
food security, it is crucial to continue elucidating the genetic basis of HS in plants. Development of
the uninucleate microspore in male reproductive stage is the most affected; therefore, more
strategies to provide resistance to HS in this process are required. Genetic and crop improvement
approaches have to be used to continue generating resistance varieties. New approaches, such as
direct mutagenesis are promising in this area.

References

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Chen Y, Müller F, Rieu I, Winter P (2015) Epigenetic events in plant male germ cell heat stress
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Harsant J, Pavlovic L, Chiu G, Sultmanis S, Sage TL (2013) High temperature stress and its effect on
pollen development and morphological components of harvest index in the C3 model grass
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Mach J (2015) Arabidopsis QTLs Associated with Reduction of Fertility in Response to Heat Stress. The
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Pressman E, Peet MM, Pharr DM (2002) The effect of heat stress on tomato pollen characteristics is
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