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Plant Science 181 (2011) 331– 341

Contents lists available at ScienceDirect

Plant Science

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biotic stress and control of grain number in cereals

udy Dolferus ∗, Xuemei Ji, Richard A. RichardsSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia

r t i c l e i n f o

rticle history:eceived 31 March 2011eceived in revised form 26 May 2011ccepted 26 May 2011vailable online 1 June 2011

a b s t r a c t

Grain number is the only yield component that is directly associated with increased grain yield inimportant cereal crops like wheat. Historical yield studies show that increases in grain yield are alwaysaccompanied by an increase in grain number. Adverse weather conditions can cause severe fluctuationsin grain yield and substantial yield losses in cereal crops. The problem is global and despite its impact onworld food production breeding and selection approaches have only met with limited success. A specific

eywords:erealbiotic stressrain numberollen

period during early reproductive development, the young microspore stage of pollen development, isextremely vulnerable to abiotic stress in self-fertilising cereals (wheat, rice, barley, sorghum). A betterunderstanding of the physiological and molecular processes that lead to stress-induced pollen abortionmay provide us with the key to finding solutions for maintaining grain number under abiotic stress con-ditions. Due to the complexity of the problem, stress-proofing our main cereal crops will be a challenging

terility task and will require joint input from different research disciplines.© 2011 Elsevier Ireland Ltd. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3321.1. Abiotic stress and reproductive development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3321.2. Mechanisms for abiotic stress avoidance and tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3321.3. The importance of timing: different effects at different stages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

2. Genetic improvement in tolerance to abiotic stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3332.1. How to screen for abiotic stress tolerance? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3332.2. QTL mapping and marker-assisted breeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3332.3. Breeding priorities and choice of germplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3352.4. Unravelling the genetic complexity of abiotic stress tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335

3. Abiotic stress and plant reproductive development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3353.1. Pollen formation: the Achilles tendon of reproductive development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3353.2. Why the male gametophyte? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3353.3. Tolerant germplasm is available . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3363.4. Anther sink strength and abiotic stress tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3363.5. Male sterility and pollen development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337

4. Is it all in the hormones?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3374.1. ABA: the stress hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3374.2. Importance of adaptation to abiotic stress in reproductive tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3374.3. A common regulatory pathway for abiotic stress tolerance?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337

5. Future directions of reproductive stage abiotic stress research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3385.1. Use of alternative screening methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3385.2. Markers or transgenic approaches? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3385.3. The benefits of using a model system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338

5.4. Can we fix it? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +61 2 62465010; fax: +61 2 62465000.E-mail address: rudy.dolferus@csiro.au (R. Dolferus).

168-9452/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.plantsci.2011.05.015

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339

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32 R. Dolferus et al. / Plant

. Introduction

.1. Abiotic stress and reproductive development

Maximising crop growth and yield in regions where there is abi-tic stress, which are variable in their timing and unpredictable inheir intensity, is a challenge to all plant scientists engaged in thisrea of research. The variability in timing and intensity of stresseans that there is no straightforward path towards understanding

he molecular and physiological basis of growth and yield forma-ion. To make progress in understanding the physiology of stressesponses and then translate this to develop a molecular under-tanding and fully progress towards breeding, it is important todentify the most vulnerable aspects of growth and yield formationnd focus on it. Plants are opportunistic; their genetic and devel-pmental program is tuned by environmental signals. Substantialesearch has been conducted on the effect of abiotic stress dur-ng early crop growth and on survival following stress. However,he effect of abiotic stress on cereal reproductive development haseceived comparatively less attention, despite the dramatic effectn grain yields. Even mild abiotic stress can irreversibly affect grainield, without affecting survival of the vegetative plant parts. Therere numerous studies in cereals that have dealt with the effect ofost-anthesis stress on grain-filling and grain size [1,2], but rel-tively few studies have addressed the effect of stress on grainumber prior to flowering. Yet, it is loss in grain number, ratherhan a reduction in grain size, that largely accounts for crop yieldeduction when abiotic stress occurs. Grain number is the most vul-erable yield component in grain crops, yet very little is known of

ts control. In an evolutionary context it has made sense that underdverse conditions our crop progenitors abort important organsuch as tillers and florets so that a small number of large seeds areormed to assure some fecundity. This strategy can be importantor species in the wild but it is likely to put a limit on grain yieldn crops. The importance of grain number rather than grain size islso evident from historic yield studies showing that breeding hasuccessfully increased yield. In all cases it has been grain numberhat has been the primary determinant of yield increases and grainize is either unchanged or reduced [3]. This has occurred in bothavorable and unfavorable environments. Part of the explanationor this could be that an adequate grain size must be maintainedor market acceptance and hence more selection pressure has beenn size rather than number of grains. A result is that little is knownbout the regulation of grain number, especially for crops grown indverse conditions. The 10–15 days period before anthesis is mostmportant for the determination of grain number [4,5]. At this stagehe reproductive structures are still small and protected by the leafheaths, making them less accessible for investigation using non-estructive methods. This paper will focus only on the effect ofbiotic stress on cereal reproductive development and how thisffects grain number.

.2. Mechanisms for abiotic stress avoidance and tolerance

Reproductive development starts with the transition of a vege-ative meristem into a floral meristem and includes developmentf the floral and reproductive structures, formation of the male andemale gametophytes, fertilisation and finally seed developmentFig. 1). The timing of the switch from vegetative to reproduc-ive development is controlled by environmental cues such ashotoperiod (long/short day length) and temperature (vernalisa-ion) [6,7]. Adjustment of flowering time is therefore an essential

daptation mechanism to the expected environmental conditionss it can result in the avoidance of abiotic stress such as frost,eat and drought in a given environment. Breeding for short- or

ong-duration varieties is therefore a common breeding strategy

e 181 (2011) 331– 341

to maximise yield and adaptation to specific environments. But,avoidance mechanisms may limit yield potential when conditionsare favorable as the crop duration may be too short to achieve maxi-mum yield. Furthermore, avoidance mechanisms do not protect thecrop when unexpected abiotic stress events occur. This is particu-larly true if stress events are short and transient (e.g., cold, heator drought spells). In this case, a tolerance mechanism is requiredto protect reproductive development and guarantee maximumgrain formation. Under field conditions, it is hard to discriminatebetween avoidance and tolerance mechanisms. Screening for tol-erant germplasm in the field has most often resulted in selectionof stress-avoiding lines with an altered flowering time rather thanlines with genuine stress-tolerance.

1.3. The importance of timing: different effects at different stages

While abiotic stress affects grain size from anthesis onwards,grain number is determined in the period before anthesis (Fig. 1).The potential grain number is fixed early during spike differen-tiation. Abiotic stress affects the number of spikelet and flowerinitials that are formed on the spike. The potential grain numbercan still be further adjusted during stress situations, as develop-ing florets can abort prematurely later in development (Fig. 1). Thiscan be easily observed in rice; the rice panicle has an open structurecompared to the compact structure of wheat and barley ears, mak-ing it easier to assess the effect of stress on grain number (Fig. 2).From meiosis onwards, reproductive development becomes irre-versibly committed to male and female gametogenesis. Meiosisoccurs approximately at the same time in the ovary and anther toform the male and female gametophyte [8]. The grain number thatwas fixed before meiosis can be dramatically reduced when abioticstress coincides with meiosis and the first stage of gametogenesis.In self-fertilising cereals such as rice and wheat, cold and droughtstress at the young microspore stage of pollen development havethe highest impact on grain number as a result of pollen sterility(Fig. 2) [9,10]. Conversely, the ovary is much more resilient to theeffect of abiotic stress [10,11].

Grain number can also be affected later when abiotic stress coin-cides with anther dehiscence (Fig. 1). Failure to release pollen fromthe anther locules prevents pollination in self-fertilising speciessuch as rice and wheat. Longer stress events that overlap with theyoung microspore and dehiscence stage are expected to show thecumulative effect of both stages. Therefore, sterility that has beenattributed to stress at anthesis may actually have been caused at theyoung microspore stage of pollen development. Pollen viability inself-fertilising cereals is very short, further preventing the chance ofcross-pollination. Flowering in rice and wheat is not synchronisedand proceeds from the top of the panicle to the bottom in rice, orfrom the middle of the ear towards the top and bottom in wheat.This asynchrony in flowering provides limited protection to shorterstress events. The length and timing of the stress period can there-fore determine the number of florets that abort. In contrast, themaize ovary was shown to be most sensitive to reproductive stagedrought stress [12]. Maize is different from temperate cereals dueto the physical separation of male and female flower structures andis therefore less dependent on self-pollination than self-pollinatingcereals.

The stage-specificity (timing), combined with the length andseverity of a stress event during reproductive development deter-mines the severity of the reduction in grain number. It is unclearwhether all these events that lead to reduction in grain number areunder the same or different (stage-specific) genetic controls. Focus-

ing our understanding on the most sensitive stage of reproductivedevelopment (young microspore stage of pollen development) mayresult in reducing the impact of abiotic stress and provide obviousadvantages for germplasm and population screening and therefore

R. Dolferus et al. / Plant Science 181 (2011) 331– 341 333

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or breeding. However, once a genetic control mechanism has beendentified, its contribution to tolerance at other stages of reproduc-ive development will need to be assessed, as well as its overallffect on yield when there is no stress.

. Genetic improvement in tolerance to abiotic stress

.1. How to screen for abiotic stress tolerance?

Screening for tolerance to abiotic stress under field conditions isotoriously difficult due to variability in severity, timing and dura-ion of the stress. In the field, plants usually experience severaltresses at the same time (e.g., nutrient stress, pathogens), resultingn screening for the cumulative effect of different stresses and thenteraction between these stress responses [13]. In addition, vari-bility within populations for such factors as flowering time, planteight, water use and rooting depth further confound results. This is

particular problem in mapping populations. Attempts have beenade to control the timing and incidence of stress conditions (e.g.,

ainout shelters for drought, variation of sowing time for cold andeat stress, deep-water irrigation for cold treatments in rice, arti-cial heat and flooding treatments, etc.), but treatments are often

onger than necessary to allow capture of a maximum number oflants going through a particular stage of development resulting

n differential levels of stress applied to the population. Further-ore, these managed conditions do not overcome other variability

n populations, particularly in flowering time, that may mask theritical variation and therefore the critical QTL identification.

For the identification of genomic regions responsible for grainumber a solution is to develop a fast and effective screeningethod under managed conditions that focuses on a particular

tage of reproductive development such as the most sensitiveoung microspore stage. To achieve this, it is necessary to movecreening from the field to a controlled environment infrastruc-ure and develop more accurate, stage-specific screening methodsor germplasm discrimination. Screening approaches in controllednvironments can exclude most of the variability that cannot be

ontrolled under field conditions, but it can also introduce newhallenges that need to be carefully considered in phenotypingpproaches [14]. These screening methods may require specialised,xpensive growth facilities that allow screening of larger numbers

ct of abiotic stress on different stages of reproductive development.

of plants (e.g., populations) under accurately controlled condi-tions (temperature, humidity, lighting). In addition, since screeningincludes the flowering stage, plants will need to spend their entiregrowth cycle in these facilities, as opposed to the shorter periodsrequired for vegetative stage screenings. However, this may notneed to be the case as plants can be moved at a critical growthstage into the managed stress condition and then after the stressmoved back to the original conditions. Ultimately, field evaluationwill remain essential to demonstrate the tolerance of selected linesand the value of the gene/QTL for stress tolerance. This is more eas-ily done using near-isogenic lines (NIL) or populations differing inthe trait, gene or QTL [15]. We are currently carrying out QTL map-ping using young microspore-stage specific screening methods forcold tolerance in rice and for drought- and shading-tolerance inwheat. As an easier alternative to drought stress for QTL mappingwe are using osmotic stress [14,16]. Applying osmotic stress usinga hydroponics system makes it possible to apply the stress at theyoung microspore stage and to apply similar levels of stress to alarge population of plants – something that is impossible to controlusing soil drought conditions.

2.2. QTL mapping and marker-assisted breeding

A major culprit for the lack of success in QTL mapping forreproductive stage abiotic stress tolerance has been the screeningprocedure [13] and the confounding factors we highlight above.A detailed account of the problems generally associated with QTLmapping for abiotic stress tolerance in crops has recently beenpublished [13,17]. QTL mapping experiments have often led toidentification of multiple QTLs each with minor contributions to thestress-tolerance phenotype. It is not clear whether this is a directconsequence of the screening method (e.g., covering a multitudeof developmental stages), the inherent complexity of the geneticcontrol mechanisms or a combination of both.

When screening procedures have been optimised QTL mappingapproaches have led to major breakthroughs in the case of sub-mergence tolerance in rice [18] and salt tolerance in wheat [19],

resulting in identification of candidate genes and ultimately a bet-ter understanding of the molecular and physiological basis of thetolerance mechanism. Once isolated, candidate genes can be used intransgenic approaches to improve reproductive stage abiotic stress

334 R. Dolferus et al. / Plant Science 181 (2011) 331– 341

Fig. 2. Abiotic stress and grain number control in cereals. (A) Grain number is strongly affected by abiotic stress at the young microspore-stage. The figure shows the effect atthe young microspore stage of cold (12 ◦C, 4 days) in rice (left) and drought stress (no water for 5 days) in wheat (right). Control and stress-treated inflorescences are shownon the left and right respectively. (B) Comparison of the inflorescence of rice and wheat. The rice panicle consists of primary and secondary branches (inset), each containingspikelets. The rice panicle has a determinate growth, with terminal spikelets at the end of each branch. The wheat ear has a much denser structure. Spikelets (inset, bottomright) are arranged on a central rachis (inset, top right). The spikelets have an indeterminate growth, with only 2–4 florets developing into a grain per spikelet. (C) Abortionof grain number in rice can be caused by premature abortion of spikelets before reaching meiosis. This is easy to discriminate from abortion caused at the young microsporestage.

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olerance in other crops or in other varieties of the same crop.uch breakthroughs have so far not been made for traits during theeproductive stage for a variety of reasons. A major reason has beenhat the underlying determinants of grain number under abiotictress are largely unknown. This results in the absence of an effec-ive selection target other than final grain number. Another reasons that, for convenience, existing mapping populations developedor other traits are typically used. These are unlikely to be impor-ant for gene discovery. Instead, populations specifically developedrom tolerant and sensitive parents are required.

.3. Breeding priorities and choice of germplasm

For commercial reasons, breeding programs are inherentlyocused on factors such as high yield potential and grain qualitynd not on insurance against stress, which may or may not occur.hile many landraces seemingly have better abiotic (and biotic)

tress tolerance than commercial germplasm, they have too ofteneen excluded from breeding programs because of their negative

mpact on yield and grain quality and because of the difficultiesf trait selection. Landraces and wild ancestors have graduallytarted to find their way into breeding programs for essential traitsuch as cold tolerance in temperate climate rice [20,21], and syn-hetic wheat lines have become a valuable resource for abiotictress tolerance traits [22]. Other approaches that may also leado trait understanding and gene discovery may come from muta-enised populations [23] or from the study of model species suchs Brachypodium [24], or from drought-tolerant species found inhe indigenous flora – for example from dry regions. However,ittle is known about reproductive mechanisms under stress of

odel species or of the indigenous flora found in dry environments.deally, abiotic stress tolerance will need to be confirmed through-ut the breeding process of new cereal varieties. But the lack ofnowledge of the most critical traits, the lack of reliable screen-ng methods, the inherent genetic complexity and the absence oflosely linked genetic markers creates a stumbling block to take fulldvantage of new genetic resources. It will also need to be estab-ished whether there are interactions between specific traits forbiotic stress tolerance and other desirable traits. For instance, its not known whether the control of grain number under normalnd stress conditions actually involves the same genes or regula-ory processes and whether expression of these genes is affectedy abiotic stress. High-yielding varieties are often selected underlose-to-ideal growing conditions. It is possible that this selectionrocess has affected the sensitivity or capacity to respond to abi-tic stress. Addressing these questions will unavoidably require aetter molecular understanding of grain number control in cereals.

.4. Unravelling the genetic complexity of abiotic stress tolerance

The genetic complexity of reproductive stage abiotic stress tol-rance may be something that we need to accept and exploit tonravel the genetic basis of grain number control in cereals. Pro-ided that a focused and reliable screening method is available, QTLapping may be the method of choice to study the genetic andolecular basis of abiotic stress tolerance. One of the few known

enes that regulate grain number in cereals was identified via QTLapping: the rice Gn1 gene encodes the cytokinin catabolic enzyme

ytokinin oxidase [25]. However, it remains unknown whether Gn1xpression is affected by abiotic stress and what other genes arenvolved in relaying the cytokinin signal to the grain developmentathway. Mutagenesis is also a technique that could be considered:

election for maintenance of grain number under stress conditionsn a mutagenised stress-sensitive population could be used as phe-otype to identify mutants (or revertants, in case abiotic stressensitivity is due to a loss-of-function mutation that has been inad-

e 181 (2011) 331– 341 335

vertently selected) and then identify the corresponding gene. Theavailability of genome sequences for most of the important cerealswill greatly facilitate the identification of candidate genes and com-parative genomics can be used to match the genomic sequences ofdifferent cereal species. A better understanding of the molecularand physiological basis of abiotic stress tolerance will allow tar-geting genes within QTL regions and subsequently using TILLING(Targeting Induced Local Lesions IN Genomes) [26] to identify andcharacterise the phenotype of natural or induced mutations in thesecandidate genes.

3. Abiotic stress and plant reproductive development

3.1. Pollen formation: the Achilles tendon of reproductivedevelopment

Abiotic stress has its biggest impact on grain number whenit occurs during the early stage of pollen development. Droughtstress has long been known to affect pollen development in wheat[27,28]. An increased frequency of out-crossing is associated withpoor grain set in drought-stressed wheat crops [3]. Male game-tophyte sterility in wheat is induced even under moderate waterstress conditions [29]. Both drought stress in wheat and cold stressin rice inflict their highest levels of sterility at the time the tetradsseparate after meiosis when forming the uni-nucleate microspores[9,10]. Similarly, greatest sensitivity to cold in sorghum occurredbetween the pollen mother cell stage and the leptotene stage ofmeiosis, yet female fertility was not affected [30]. Drought stressoften occurs together with heat stress in the field and the regula-tory system for both stresses may have co-evolved [31]. Heat stressin cereals affects both pollen dehiscence and grain-filling [32,33],but a stage of high sensitivity responsible for loss in grain num-ber occurs before the ear or panicle emerges from the leaf sheath[34]. Low light conditions (shading) before anthesis also reducesgrain number in wheat [35]. This problem is due to pollen steril-ity induced at the young microspore stage and again the ovaryshowed higher resilience to stress, as measured by their ability tobe fertilised by pollen from unstressed plants [36]. Interestingly, inthe absence of stress conditions, high nitrogen levels boost grainnumber but have an adverse effect on pollen fertility and abioticstress tolerance such as cold in rice [37]. The high sensitivity ofyoung microspore-stage pollen to a variety of abiotic stress pro-vides opportunities for germplasm screening. Pollen fertility andsterility are also distinct phenotypes that provide an entry point todetermine the molecular basis of the problem.

3.2. Why the male gametophyte?

The higher sensitivity of male gametophyte development to abi-otic stress may have something to do with the unique properties ofthe innermost layer of the anther wall, the tapetum. The tapetum inanthers is to some extent similar to the placenta in mammals; it is ahighly specialised sporophytic secretion cell layer that is dedicatedto feeding the nascent microspores. After fulfilling this function thetapetum undergoes a natural, pre-programmed cell death responseand its cellular content is consumed by the young microspores(Fig. 3). This nursing function of the tapetum takes extreme propor-tions in plants with an amoeboid tapetum, where the microsporesare completely engulfed by the tapetum layer after meiosis [38,39]The tapetum deposits the intine and exine layers of the pollencell wall and it secretes the locule fluid which contains nutrients

for pollen development [40,41]. During meiosis and at the youngmicrospore stage the tapetum is metabolically extremely active andthe anther is the floral organ with the highest sink strength [42].To satisfy the high energy requirements, the number of mitochon-

336 R. Dolferus et al. / Plant Scienc

Fig. 3. Cross sections of rice anthers at the early stage of male gametogenesis. Thetop picture shows an anther just before meiosis. The anther wall consists of fourlayers. From outside to inside, these layers are the epidermis (Ep), the endothecium(En), the middle layer (M) and the tapetum (T). In the centre are the meiocytes(Me) that will undergo meiosis and give rise to the microspores. The bottom pictureshows an anther cross section at the late uni-nucleate stage. The middle layer hasdegenerated and young microspores are still attached to the tapetum layer (darkbp

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lue layer) which is now reduced in thickness and will disappear completely towardsollen maturity.

ria per tapetum cell is increased 20–40-fold [43]. The exact timingf the cell death response in the tapetum is also critical for polleniability. Both cold and drought stress have been shown to cause

pre-mature cell death response in the tapetum [44,45], suggest-ng that abiotic stress interferes with the important functions ofhe tapetum. In tropical grasses such as maize, the nucellus is sup-orted by the pedicel. The placento-chalaza cell layer separates theucellus from the pedicel. Under drought conditions, the placento-halazal cells undergo a senescence response, thereby restrictingugar flow to the nucellus [46]. This situation is reminiscent of theapetum and may explain why the maize ovary is highly sensitiveo drought stress.

.3. Tolerant germplasm is available

The identification of tolerant and sensitive germplasm for repro-uctive stage stress tolerance should allow the introduction of the

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trait into commercial germplasm. Comparing the response of tol-erant and sensitive germplasm will also enable us to identify themolecular basis of the tolerance mechanism. A screening methodbased on the high sensitivity of young microspore-stage pollendevelopment was used to identify cold-tolerant rice germplasmin a collection of rice varieties from the Yunnan province in China,where rice is traditionally grown at high altitudes (2000–2400 m)[9]. A high altitude sorghum line also had a strong tolerance at theyoung microspore stage [47]. A wheat line derived from a CIMMYTsynthetic wheat was shown to have strong young microspore-stage drought tolerance [10]. These examples show that abioticstress tolerance is present in cereals where the trait may have beenmaintained out of necessity, often imposed by topographic circum-stances (e.g., high altitude rice for cold-tolerance). This observationraises the question whether tolerance mechanisms may have beeninadvertently lost from current commercial germplasm, eitherthrough direct/indirect selection or due to negative selection dragwhile selecting for other traits. For example, in wheat germplasmfrom CIMMYT which has been selected under irrigated conditionshas been very successfully adopted in semi-arid countries such asAustralia. This may have resulted in the loss of tolerance in currentAustralian wheat cultivars [48]. Genes responsible for maintenanceof grain number under drought may also have been lost due toselection pressure in breeding programs for grain size and hecto-liter weight which is demanded by industry. If there is a terminaldrought then genotypes that are conservative in number of grainsthey set may be selected as these are also likely to have larger grainsat maturity.

3.4. Anther sink strength and abiotic stress tolerance

The investigation of the effects of cold and drought stress onthe young microspore stage of pollen development revealed thatthere is a major difference in the control of sink strength betweentolerant and sensitive germplasm in both rice and wheat [9,10].Plasmodesmata between the tapetum and the outer cell layers ofthe anther wall disappear at meiosis, and callose deposition isolatesthe meiocytes from the rest of the anther. Uptake of sugars into thetapetum therefore has to occur via the apoplast, requiring an activeuploading process involving cell wall invertase and monosaccha-ride transporters [49,50]. While sensitive germplasm has reducedtapetum cell wall invertase levels under cold and drought, toler-ant germplasm maintains sink strength [9,10,51]. Subsequently, apremature cell death response is observed in the tapetum of sen-sitive varieties, leading to abortion of pollen development [44,45].Depletion of sucrose and starch was also observed in tomato andcapsicum pollen following heat stress [52,53] and heat stress affectscell wall invertase activity in sorghum pollen [54,55]. A critical dif-ference between tolerant and sensitive germplasm is the capacityto control and maintain sink strength and sugar supply to sustainpollen development. The regulatory pathway that controls anthersink strength and cell wall invertase activity remains unknown.A second peak in sink strength occurs during flowering and grainfilling and cell wall invertase is known to be equally importantin this step [56,57]. In drought-stressed maize ovaries, cell wallinvertase activity is repressed in the placento-chalazal cell layer[46]. The mechanism of ovary abortion under drought conditions inmaize may be similar to cold- and drought-induced pollen abortionin rice and wheat. However, drought-tolerant wheat germplasmthat is able to maintain sink strength when stressed at the young

microspore-stage fails to maintain sink strength when stressedduring anthesis and grain-filling [10]. This indicates that the geneticcontrol of sink strength is different during pollen and grain devel-opment.

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.5. Male sterility and pollen development

Cytoplasmic male sterility (CMS) in plants is a gain-of-functionutation caused by complex rearrangements in essential mito-

hondrial genes. These mitochondrial dysfunctions only affect thenthers and not the other plant parts. Some of the CMS mutationsre known to disturb mitochondrial metabolism in the tapetumf anthers. This may result in poor respiratory performance andn inability of the mitochondria to meet energy requirements foruccessful gametogenesis [58,59]. The result is pre-mature tapetalell death and abortion of pollen development. The mitochondrialay an important role in programmed cell death and CMS haseen important in demonstrating the role of plant mitochondria inMS [59]. Although CMS and abiotic stress at the young microsporetage in anthers both trigger an accelerated programmed cell deathesponse in the tapetum, the cell death response may be a conver-ence point of two totally different upstream triggering pathways.MS has been exploited in plant heterosis breeding for the pro-uction of hybrid seed. An alternative method that is simpler toeverse is the use of gametocides or chemical hybridisation agentsCHA) [60,61]. A wide variety of chemicals have been used to target

ale fertility at various stages of anther development. Althoughome of these components have been shown to interfere withollen meiosis and the early stages of microspore development,

n most cases it is not clear what their cellular targets are. It isowever possible that some chemical agents, through interferenceith tapetal function, have a similar result to the effect of abiotic

tress on pollen development. Many transgenic approaches thatffect metabolism and cellular function in anthers and particularlyhe tapetum have resulted in the induction of male sterility. Thesepproaches range from the introduction of a bacterial ribonucleaseene [62] to the manipulation of genes involved in callose deposi-ion during meiosis [63] and inhibition of respiratory enzymes [64].ll these examples further illustrate the vulnerability of male game-

ophyte development and the important role the tapetum plays inontrolling pollen fertility.

. Is it all in the hormones?

.1. ABA: the stress hormone

ABA was a prime suspect as a regulator of sink strength. Inhe case of cold- and drought-induced sterility in rice and wheatBA represses cell wall invertase expression and cold and drought-

olerant germplasm does not accumulate ABA [16,65]. ABA plays role in male sterility in tomato [66] and may also be involved inesponse to heat stress [67]. Reduction in anther ABA levels dur-ng chilling of transgenic rice over-expressing the ABA catabolicene ABA 8′-hydroxylase resulted in maintenance of sink strengthnd improved cold tolerance, suggesting that regulation of ABAatabolism in anthers is important for regulating ABA homeostasis16]. ABA homeostasis in anthers is different in tolerant and sen-itive germplasm, but it remains unknown how ABA homeostasiss regulated. Cold and drought tolerant rice and wheat lines were

ore sensitive to ABA treatment of the reproductive structureshan sensitive lines [16,65]. Selection of ABA sensitivity mutantsas used to identify drought-tolerant wheat [68] and QTLs forBA sensitivity have been identified in wheat with the intention

o improve ABA-mediated abiotic stress responses [69]. In maize, aTL that affects root traits and leaf ABA concentration was shown

o affect grain yield [70]. However, using ABA sensitivity as an

ndirect selection method for abiotic stress tolerance may haveecondary effects, as ABA plays a role in many important aspectsf plant development (seed dormancy, regulation of stomatal clo-ure). Although increased ABA levels are negatively correlated with

e 181 (2011) 331– 341 337

abiotic stress tolerance at the reproductive stage, the oppositeappears to be true at the vegetative stage. There is plenty of evi-dence that ABA treatment at the vegetative stage can improveacclimation to a variety of abiotic stress conditions [71–74]. Thecold and drought-tolerant lines we are currently investigating inrice and wheat are not noticeably disadvantaged at the vegetativestage. ABA has different roles in the vegetative and reproductiveplant parts. Unlike the vegetative plant parts, the reproductive partsare sink tissues for sugars. Regulation of ABA homeostasis is likely toinvolve interaction with other plant hormones that regulate plantdevelopment (auxin, cytokinins, gibberellic acid, ethylene, . . .) andthese interactions may function differently in the reproductive andvegetative tissues.

4.2. Importance of adaptation to abiotic stress in reproductivetissues

While ABA in anthers plays a role in controlling sink strengthand sugar supply to the young microspores, the response to abioticstress also involves ABA-independent components. In the case ofcold and drought stress, the ABA-independent response is regulatedby the CBF/DREB1 family of transcription factors and is thought toactivate the acclimation and adaptation response that allows longerterm survival of abiotic stress conditions [75–77]. However, thefact that ABA treatment can improve adaptation to abiotic stresssuggests that the two pathways are not strictly independent. Anal-yses of abiotic stress responses using various “-omics” technologiesrevealed that plants activate a variety of defence responses to pro-tect the cellular machinery against damage [78]. These adaptationsinclude synthesis of lipids to protect membranes, antifreeze pro-teins that protect against frost, osmotic components to maintainwater status in the cell (e.g., sugars such as trehalose and fruc-tans), chaperone proteins that protect protein folding and a varietyof other protective components (e.g., proline, polyamines) [78].However, a dangerous side-effect of all abiotic stress conditionsis the overproduction of reactive oxygen species (ROS), resulting inoxidative stress and irreversible damage to various cellular compo-nents. Plants activate an antioxidant scavenging mechanism suchas the ascorbate-glutathione pathway to inactivate ROS [79,80]. Theoxidative stress response in particular shows that adaptation to var-ious abiotic stress situations in plants is shared by different typesof environmental injuries. Little is known about the adaptation toabiotic stress at the reproductive stage, but it is expected that ametabolically very active and vulnerable cell layer such as the tape-tum will benefit from some of these protection mechanisms againststress. Adaptation responses to abiotic stress have so far been stud-ied in model plants or crop plants with unknown ranking in termsof stress tolerance/sensitivity. Our rice and wheat lines with knowntolerance and sensitivity to cold and drought will allow investigat-ing the quantitative and qualitative differences in the adaptationresponse between tolerant and sensitive germplasm.

4.3. A common regulatory pathway for abiotic stress tolerance?

The similarity that exists between the effects of abiotic stresson pollen development suggests that there may be an overlapin the regulatory pathways that control stress tolerance (Fig. 4).It has been shown that there is a great deal of overlap betweenthe genes that are recruited by both cold and drought stress inplants [81]. This is an important observation, as it may providethe basis for alternative screening methods for identifying tolerantgermplasm. Tolerance genes that play a role in response pathways

that overlap between different stresses are expected to provideprotection against various stresses (Fig. 4). One of our drought-tolerant wheat lines (Halberd) is also tolerant to heat stress [10,82]and cold-tolerant rice is also more tolerant to reproductive stage

338 R. Dolferus et al. / Plant Scienc

Cold Drought Others

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GrainNumber

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Tolerance?

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Fig. 4. Effect of abiotic stress on pollen development. Cold, drought and other abi-otic stress conditions (heat, salinity, shading, . . .) induce a number of responses(arrows) in reproductive organs such as anthers. Some of these responses are spe-cific but others (indicated with asterisk) are shared by different stresses. The controloas

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f pollen fertility appears to be part of a stress response that is shared by differentbiotic stress conditions: tolerance to drought stress can cross-protect against othertresses such as cold and shading conditions.

rought stress (Dolferus, unpublished result). In addition, ourrought-tolerant germplasm is also shading-tolerant [36]. Alterna-ive screening methods may therefore provide a powerful way todentify QTLs and genes for an abiotic stress tolerance mechanismhat is shared between different stress responses.

. Future directions of reproductive stage abiotic stressesearch

.1. Use of alternative screening methods

Conventional breeding and selection has been very successful inteadily increasing crop yields, even without having knowledge ofhe underlying traits. However, breeding in stress prone environ-

ents is challenging and progress is slow. This is primarily due tohe high degree of seasonal variability in temperature and rainfall,esulting in large genotype x environment interactions. In ordero further improve abiotic stress tolerance in cereals it is neces-ary to increase our knowledge of the current bottlenecks thatonstrain yield [15]. In the case of the control of grain numbere require a better understanding of cereal reproductive devel-

pment, how it is affected by stress and what are the underlyingechanisms responsible for the variation in grain number. Reli-

ble screening methods are a major bottleneck for improvement ofeproductive stage stress tolerance. A better understanding of theffect of stress on reproductive development will lead to develop-ent of more reliable screening alternatives that are targeted to

he very root of the problem. The similarity that exists betweenifferent abiotic stress conditions that cause pollen abortion mayllow us to use alternative stress treatments. This may be particu-arly useful for difficult to control stress treatments such as drought.smotic stress treatments using hydroponics and polyethylene gly-ol as osmoticum have been used in the past to impose watertress conditions [14,83]. However, it is also possible to replacerought stress with an easier to apply abiotic stress that has theame effect on pollen development (e.g., cold, shading). We haveecently shown that drought-tolerant wheat germplasm is also tol-rant to shading conditions [36], suggesting that we could replacerought stress treatment by a more simple shading treatment athe young microspore stage. The QTL mapping work that we areurrently carrying out in wheat for osmotic stress and shading will

erve as a test case to prove the validity of this approach. Oncehe knowledge about the molecular and physiological basis of theroblem has progressed to the stage where we can target specificomponents of the abiotic stress signalling pathway we may be able

e 181 (2011) 331– 341

to design alternative screening methods based on chemical treat-ments (e.g., hormones or hormone inhibitors, signal transductioninhibitors, . . .). Although alternative screening methods could ini-tially facilitate the screening of large population for QTL mapping –and make this process more reliable – the ultimate test will remainthe confirmation of selected material under field conditions. QTLmapping and marker identification will therefore require the inputand close collaboration between various research disciplines.

5.2. Markers or transgenic approaches?

The identification of candidate genes is critical for improvingour understanding of the molecular and physiological basis of abi-otic stress tolerance. These genes, together with the knowledgeof how they fit into the tolerance pathway will enable us to usetransgenic approaches to improve abiotic stress tolerance in cerealcrops. Transformation methods have been established for cerealsbut they may not work with all elite breeding lines. However, trans-genic approaches that target abiotic stress tolerance will need to becarried out judiciously, considering the fact that many manipula-tions of genes that interfere with tapetum metabolism and functionhave resulted in male sterility (see above). The choice of promoterto drive expression of the transgene will also be critical. Con-stitutive promoters that express the transgene ectopically in allplant parts may lead to negative side-effects and compromise plantyield. This was illustrated using the CBF/DREB1 transcription fac-tors that lead to improved vegetative cold and drought-tolerance,but also stunted plant growth [84,85]. CBF/DREB1 transcription fac-tors are normally expressed only in the vascular parenchyma cells[86]; they are normally induced by stress conditions and there isobviously a penalty associated with expressing a stress adaptationresponse all the time and in all plant parts. An attempt to reduceanther ABA levels by expressing the ABA catabolic gene ABA 8′-hydroxylase using a strong tapetum-specific promoter has led toreduced anther ABA levels, improved sink strength and improvedcold-tolerance in rice [16], suggesting that transgenic approachesare indeed feasible. The availability of candidate genes for stress tol-erance in reproductive structures will enable us to design perfectmarkers that can be used for marker-assisted breeding.

5.3. The benefits of using a model system

The availability of a model plant system with a small, uncom-plicated and sequenced genome like Arabidopsis thaliana has beencritical in advancing our general knowledge about plants. Rice isgenerally considered as a model for cereal research, but Brachy-podium has been proposed as a model for the Triticeae cerealsbecause of its phylogenetic position between rice and wheat.The advantage of Brachypodium over rice is a smaller genomesize (advantage for mutagenesis), shorter generation time, simplergrowth requirements and easy/faster transformation [24]. Brachy-podium is also the only wild grass with a sequenced genome,allowing us to assess the effects of domestication based on co-linearity with other cereal genomes [87]. Unfortunately for rice, acrop plant that was domesticated more than 10,000 years ago, wehave the genome sequence of the two most commonly grown sub-species (indica and japonica) but not the blue-print sequence ofthe original wild rice genome (Oryza rufipogon). Brachypodium maynaturally be more resilient to abiotic stress compared to domesti-cated cereals, making this wild grass less suitable to study the effectof abiotic stress on grain production. The huge size and hexaploidnature of the wheat genome will always be an obstacle to make

fast progress, even with an available genome sequence. But impor-tant genetic variability for reproductive drought tolerance has beenidentified in wheat [10]. Rice is still the most developed cerealresearch model and there are many tools to mine the available

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enome sequence. Rice is a diploid species with a relatively smallenome and tools are available to mine the genome information.n addition, as mentioned earlier, rice offers advantages comparedo other cereals for studying grain number control by abiotic stressFig. 2). The co-linearity that exists between the rice and wheatenome sequences will make it possible to identify wheat genesased on known genes or map positions in rice (synteny mapping),n approach which has already proven its merits [88,89]. Usinghe combination of rice and wheat will be useful for map-basedloning and identification of candidate genes for reproductive stagebiotic stress tolerance. A laborious and time-consuming part ofne-mapping is the development of large populations to fine-ap the QTL regions, especially the identification of polymorphicTL-region-specific markers [90,91]. This requires large targetedopulations (e.g., NILs) to provide the genetic resolution requiredor fine-mapping QTLs to a mendelian factor. This approach canlso benefit from meta-analysis to improve resolution of QTLs [92].he availability of SNP (single nucleotide polymorphism) markersn recent years has facilitated this work. Although SNP markers arevailable for both rice and wheat, the rice SNP platform is partic-larly well developed and can provide resolution to the gene level93,94]. Knowledge about the molecular and physiological basis ofeproductive stage abiotic stress tolerance will also help to identifyotential candidate genes within QTL regions and design gene- orllele-specific markers for these genes based on the rice genomennotation and SNP information. TILLING [26] can then be used as

reverse genetic approach to identify mutants in candidate genesnd study the effect of these mutations on abiotic stress tolerance.ILLING is a technique that has been shown to work well in bothice and wheat [95,96].

.4. Can we fix it?

Breeding and selection over decades have dramaticallyncreased crop yield and grain marketability of all cereals inoth favorable and unfavorable conditions. Increases in yield havelways been associated with an increase in grain number whereasrain size is generally stable. Most of the increase in grain numbernder favorable conditions has been associated with an increase

n partitioning of carbon to the developing reproductive structuresust before flowering. This additional carbohydrate has probablyome from the reduction in stem growth in semi-dwarf cereals97]. Corresponding flow-on effects of yield increases to less favor-ble conditions where abiotic stress is not severe has occurred. Thisnowledge has resulted in breeders being able to target specificenes associated with semi-dwarf stature in wheat and rice and itas resulted in a greater grain number and yield. However, knowl-dge of the causes of a reduction in grain number in the presencef abiotic stress such as drought, low and high temperature aretill rudimentary and further research is required to better under-tand the fundamental processes responsible for sterility. Also, its important to understand whether these processes are commoncross different abiotic stress conditions and to understand the hor-onal and metabolic changes responsible. With this knowledge

etter screening methods will be developed, improved parentalaterial discovered and ultimately genomic regions and the key

enes involved will be identified. This in turn will lead to moleculararkers and fast and efficient screening methods for use by breed-

rs in their target environments. It will also help answer questionsbout the specific role of hormones in the determination of grainumber in the presence of stress, whether there are common genes

or tolerance to different stresses and whether intensive breeding

his century may have resulted in a loss of important genes forolerance to abiotic stress. An understanding will also be achievedbout possible trade-offs between grain number and grain size. Forxample, some stresses that reduce grain size and where water is

[

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not a limitation, there is unlikely to be a trade-off in grain number.However, where drought reduces fertility at the young microsporestage and drought is not relieved then genes that maintain fertilityare likely to be undesirable as more grains will be produced that aresmall in size and therefore unacceptable to markets. Crop simula-tion models using historic weather data for a region can be usefulhere to assess the importance of traits and possible trade-offs [98].

Our understanding of the control of grain number in cereals thatexperience abiotic stress has increased in recent times. The mostvulnerable period during meiosis is common in such diverse speciesas rice grown in the tropics and wheat grown in more temper-ate environments. Mechanisms of protection for different stresses,be they cold or drought or heat, also appear to be similar. Recentadvances in the development of fast, efficient screening methodsand the identification of highly sensitive and tolerant germplasmwithin species are expected to lead to important new discoveries infuture years. It is also expected that these advances will also shedlight on the control of grain number under favorable conditions,which in turn could lead to genetic advances in yield potential.

Acknowledgements

The research presented in this paper was supported by theAustralian Grains Research and Development Corporation (wheat;GRDC grants CSP00089, CSP00130 and CSP00143) and the Aus-tralian Rural Industries Research and Development Corporation(rice; RIRDC grant US-143A).

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