Structure of rice caryopsis in relation to strategies for enhancing yield

Structure of rice caryopsis in relation to strategies for enhancing yield

G.A.I. Ebenezer, S. Krishnan, and P. Dayanandan

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Rice varieties with higher yield potential can be developed by increasing the number of grains per area, by increasing the grain weight, or by a combina­

tion of both. Our studies on the structure, histochemistry, grain filling, and response to plant growth regulators (PGRs) have clarified certain unique fea­

tures of the spikelet of rice grains and have identified strategies required to enhance the weight of rice grains, The most important barrier to grain weight is the space limitation imposed by the fertile glumes, the palea and lemma within which the caryopsis should develop.

Experiments with intact plants and excised spikelets reveal that most PGRs, singly or in combination, do not influence the growth of glumes.

Brassinoloide (BR) promotes growth of the palea and lemma when applied during the panicle initiation stage. BR at 10-7 M and benzylaminopurine (BAP) at 10‘5 M applied together as a soaking spray increase grain size and dry weight by 39%. This is achieved through promotion of cell size and cell num­

ber, of both the palea and lemma, and the caryopsis within. The aleurone • cells of the treated grains increase to 160,000 from 75,400 in control grains.

Strategies for yield improvement could aim at increasing the dry grain weight, either by (1) breeding rice varieties with a larger palea and lemma or by (2) producing varieties that respond to PGR application by rapidly increasing the size of the palea and lemma.

A m ature rice grain varies in length betw een 5 and 10 mm, weighs between 15 and 40 mg, and has a specific gravity o f 1 to 1.2 and porosity o f 60% (Houston 1972). An IR50 rice grain at the tim e o f sow ing has a length/w idth/thickness o f 8.5/2.7/1.B mm, respectively. T he dry w eight o f the grain is 21.6 mg. The length/w idth/thickness of a caryopsis is 6.1/9.1/1.5 mm, respectively. A m ature grain consists o f a caryopsis sur­

rounded by a palea and a lem m a and two sterile lem m as (Fig. 1). The base o f the lem m a is sw ollen and is know n as the rachilla. A n abscission zone occurs below the two sterile lem m as and helps separate the pedicel from the paddy grain. The apical

109

Fig. 1. Perspective diagram of a rice grain.

port ion o f the pedicel is considered to represent a rudim entary glum e. In transverse section, the m id-region o f the caryopsis show s a typical arrangem ent o f the aleurone layer surrounding the endosperm cells. It is estim ated that about 75,400 endosperm cells m ake up th e bulk o f the storage tissue.

Grain filling and transport of assimilates

D uring early developm ent o f the caryopsis, the endosperm and em bryo are isolated from the rest o f the m aternal tissue by a prom inent cuticular layer that surrounds the nucellar epiderm is. H ow ever, nutrients are transported to the endosperm through a single ovular vascular bundle on the ventral side o f the ovary (Fig. 2). Figure 3 is a sum m ary diagram o f our current understanding o f the structure o f the vascular region involved in the transport o f assim ilates int o the developing caryopsis. A pigm ent strand and a nucellar projection m ediate this transport process. B etw een I and 7 days after fertilization, nutrient m aterial enters the endosperm through these still persistent nu­

c e lla r tis su e s. S u b se q u e n tly , so lu te s fro m th e o v u la r v a s c u la r b u n d le m o v e circum ferentially into the nucellar epiderm is and centripetally from the nucellar ep i­

derm is into the endosperm . T he nucellar epiderm is is specially reinforced with wall thickenings to provide m echanical support during this period o f active enlargem ent o f the caryopsis. A com parison o f grain filling in C 3 and C 4 cereals suggests that rice has structural features interm ediate betw een these tw o types (Krishnan 1996). The

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Fig. 2. Cross section of a mature rice caryopsls showing red autofluorescence of chlorophyll In cross cells. The cross-cell photosynthesis may also contribute to the as similale that enters the earyopsls. X 250. CO = cross cell, OV = ovular vascular bundle, I" = endosperm.

Fig. 3. Diagrammatic representation of a portion of the ventral side of the cary opsis.

Pigment stran :l Integument Nucellar projection Nucellar epidermis Cross cell Tube cell Aleurone Plasmodesmatit Protein bodies Lipid globule Amyloplast Mitochondria Endosperm

— ■— — Wall thickenings Pericarp Phloem Xylem

Structure of rice caryopsis in relation lo strategies for enhancing yield 111

.situation in l ice is allied to that o f w heat although no xylem discontinuity is known in rice. T he pigm ent strand and the nucellar projection in rice are not as well differenti­

ated as in wheat. T he significance o f all m aternal and filial structures associated with grain filling is likely to be b elter understood in the near future since the developm en­

tal biology o f the cereal endosperm is now beginning to be analyzed with tools o f m olecular biology (Olsen et al 1999).

Anatomy and histochemistry of glumes

A large num ber o f cultivars and species w ere exam ined for the organization o f grain structure. The palea and lem m a reach their m axim um length at the tim e o f anthesis.

The outer surface o f fertile glum es, palea, and lem m a consists o f rows o f large epider­

mal cells with silicified knobs alternating with long, typically bicelled hairs. The apical cell o f the bicelled hair is very thin and collapsible. A lem m a generally lias about 35 stom ata on its outer surface. T hese occur m ostly in rows along the two edges that curve around the palea. A few stom ata are also found on the m ucro and apiculus o r awn, and the bases o f sterile glum es. The stom ata on the palea are confined to the apiculus. T he stom atal apparatus on the lem m a is about tw ice as large as that on a leaf. T he guard cells o f these stom ata possess am yloplasts.

About. 11 species o f Oiyzci have been analyzed for the frequency and dim en­

sions o f the stom atal apparatus found on the lem m a. T he length o f stom a ranges from 26 pm in the case o f 0 . eichingeri to 32. p m in 0 . punctata. W idth o f the stom atal apparatus ranges from 33 to 45 pm . T he largest stom atal pore is found in (). latifolia.

Most species have stom ata in a single row. In (). grandiglum is, however, tw o rows of stom ata occur in the m iddle o f both (he curving edges o f the lemma. In O. grdnulata, stom ata occur only over the apiculus. T he inner epiderm is o f the palea and lem m a is com posed o f large cells, stom ata, and bicelled hairs. T he latter are concentrated in the apical region (Ebenczer et al 1990). B etw een the inner and outer epiderm is are three layers o f cells, tw o o f w hich, located on the side o f the outer epiderm is, develop into long, highly sclerified fibers (Figs. 4 and 5). T he layer close to the inner epiderm is

Fig. 4. Transverse section of mature lemmn showing silica knobs on the outer epidermis. X 500. Si = silica knob, OE = outer epidermis, FI = fi ber layer, IE = inner epidermis, Pa = parenchyma.

112 fbenezer et al

remains thin-w alled, and along with the inner epiderm is is com pressed during the developm ent o f the caryopsis. O nly the outer epiderm is contains chlorophyll

Spikelets containing near m ature grains were excised and placed in dye solu­

tions. The inner and outer epiderm al peels w ere then exam ined with a microscope.

Figure 6 reveals that Ihc bicelled hairs readily lake up dyes and accum ulate them in the apical cells. The Prussian blue technique reveals that w ater readily enters into the

Fig. 5. Longitudinal section of lemma.

X 420. IE = Inner epidermis, Pa = pa­

renchyma, FI = fiber layer, OF = outer epidermis.

Fig. 6. (A) Inner epidermis of lemma of Ory/n saliva eulti- var Ponnl stained with Coomassie brilliant blue R 250 showing protein within the bicelled hair. X 3.25. (B) Outer epidermis of lemma of 0. glaberrima after transport of reagents for Prussian blue staining. The microhairs are bicelled, tricelled, and four-celled. X 125. PR = protein, TH = three-celled hair.

Structure of rice caryopsis In relation to strategies for enhancing yieli I 113

bicelled hairs. Such hairs are present on both the o uter and inner surfaces o f (he palea and lem m a. Interestingly, the num ber o f cells in such hairs is not always two; thrcc- hnd four-celled hairs w ere also observed. T hese hairs also accum ulate am orphous protein as show n by staining with C oom assie brilliant blue reagent in IR50, as well as in cullivar Ponni and O. grandiglumix.

Physiology of glumes

The palea and lem m a arc fully differentiated at the tim e o f anthesis and have about I mg o f total chlorophyll per gram fresh weight. T he caryopsis and flag leaf have about 0.4 mg and 3 mg o f chlorophyll per gram fresh weight, respectively. The spikclet has very little silica before anthesis but rapidly accum ulates silica from the day o f anthe­

sis until about 20% o f the dry w eight o f the hull is m ade o f silica. W ater content o f the palea and lem m a drops from about 85% ju st before anthesis to 10% at the tim e o f grain maturity.

T he transpiration rate is very high in spikelets com pared with that o f a flag leaf.

U nder identical environm ental conditions, the transpiration rate o f the spikelet is 3.6 mg e n r 2 l r 1 versus 2 m g cm 2 I r 1 for the flag leaf. Com pared with a m axim um num ber o f about 100 stom ata c u r 2 in a spikelet, the adaxial surface o f a flag leaf has about 35,000 stom ata c u r 2. The abaxial surface o f the flag leaf has about 40,000 stom ata cm r2. W ater loss through a single stom a on the outer surface o f a lem m a is 1,200 tim es m ore than through a single stom a o f a flag leaf. A single spikelet loses about 4 mg o f w ater l r 1, w hereas an entire flag leaf transpires only 60 m g o f w ater I r 1. T his high rate o f w ater loss is probably an essential physiological requirem ent for grain filling.

Strategies for enhancing yield

T he m ost im portant b arrier to an increase in grain w eight is the space lim itation im ­ posed by the fertile glum es, the palea and lem m a, that cover the caryopsis. They provide a restricted space w ithin w hich the caryopsis should develop. M atsushim a (1970) show ed that insertion o f vinyl film inside the palea and lem m a reduced the space, resulting in a correspondingly reduced caryopsis. Takeda (1973) dem onstrated that the differential rate o f elongation in som e cultivars may produce a caryopsis longer than the length o f space provided w ithin the palea and lem m a, and that such an im balance in grow th m ay result in a notched caryopsis. It m ay not be possible, how ­ ever, to do aw ay w ith the palea and lem m a since they appear to be essential protective structures and organs that regulate the transport o f w ater and m inerals during grain filling and m aturation.

C lipping the apical 2/3 to 1/2 portion o f the glum e interferes with the normal developm ent o f ovaries, leading to dim inutive caryopsis. C overing the apical I /3 por­

tion o f the spikelet with lanolin or plastic tubes, how ever, results in the developm ent o f the caryopsis (Fig. 7), although the fruit is abnorm al in shape and incom pletely filled. T hese experim ents also reveal that the grow th potential o f the caryopsis is

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Fig. 7. The apical region of the glumes was clipped and the exposed legion cov­

ered with a plastic tube (PT).

lim ited by the space provided w ithin the palea and lem m a, thus confirm ing the previ­

ous observations o f M atsushim a (1970), Seo and Ota (1982), Takeda (1973), and Takahashi and Takeda (1971).

Rice varieties w ith higher yield potential can be developed by increasing the num ber o f grains per area, by increasing the grain weight, or by a com bination of both. B reeding strategies have prim arily focused on increasing the number o f grains, and it is assum ed that the grain-w eight com ponent is recalcitrant. A large and heavier caryopsis can be obtained only when the space enclosed by the palea and lemma is larger, thus perm itting the caryopsis to enlarge and fill the space. The palea and lemma, however, are fully developed even at the tim e o f anlhesis. These structures are gener­

ally insensitive to external application o f plant growth regulators (PG Rs) (Ebenezer 1989, E benezer et al 1990). Any attem pt to alter the size o f the palea and lemma should be carried out during the early stages o f panicle initiation and not at the tim e of anthesis.

Table 1 sum m arizes investigations on the effects o f different PGRs. The length, width, and thickness o f control grains are 8.5 m m , 2.7 mm, and 1.8 mm, respectively.

B rassinoloide (BR) + benzylam inopurine (BA P) treatm ent enhances grain size by affecting all three dim ensions (10.6 mm, 3.1 mm, and 2.2 mm). In BR- and BAP treated plants, the palea and lem m a are larger even at the tim e of anthesis. Tieatmenl also increases the num ber o f aleurone cells from 202 in the control to 280 per cross- sectional area. T he aleurone cells occur m ostly in a single layer surrounding the en­

dosperm cells. T he endosperm cell num ber also increases from 265 per cross-sec­

tional area in the control to 400 in the treated caryopsis. Careful m icroscopic analysis reveals that Ihe total num ber o f endosperm cells increases from 75,400 in the control caryopsis to about 160,000 cells in the treated caryopsis. The size o f an endosperm cell also increases by 40% in the BR + BA P treatm ent.

H istochem ical studies o f the control and BR + B A P-treated grains reveal that an increase in grain size does not alter the pattern o f distribution o f the m ajor storage

Structure of rice caryopsis in relation to strategies for enhancing yie Id 115

■ ^jl. ^{c n o c i} oi plant gtowth regulators oil yield In IR50 rice.

Treatment®

(M)

Fresh weight

of 100 %

grains (g)

Dry weight

of 100 %

grains (g)

Control 2.32

_

_

Brassinoloide (BR) 10-7 M

2.85 23.2 2.73 26.4

Benzylarninopurine (BAP) 2.71

10-e M

Glbberellic acid (GA3) 10 '° M

2.56 10.4 2.46 14.0

Kinetin (KIN) 10-5 M

2.73 17.8 2.67 23.7

BR 10-7 M + KIN 10~5 M

2.84 23.0 2.74 26.8

BR 10-7 M + BAP 10“5 M

3.12 34.7 3.00 38.9

BR 10~7 M + GA3 10-« M

2.72 17.3 2.54 17.6

SM = molar.

Source: Krlshnan et al (1999).

m aterials. Jn botli the control and treated caryopsis, aleurone cells and lipid occur m ostly in the subaleurone layers. T h e b ulk o f the endosperm is m ade up o f starch and all o ther storage com ponents are sim ilarly d istributed in both grains (K rishnan 1996).

D evelopm ent o f the caryopsis is a p ostanthesis phenom enon and can be in flu ­ enced only if B R and B A P are applied im m ediately after anthesis. Previous pot-cul- ture experim ents have show n that auxin and triacontanol-based products prom ote yield in rice. O f all the P G R s that w e investigated, B R appears to be the m ost p ro m ­ ising, especially at 10-7 M w ith 10~5 M BAP.

O ther im portant strategies for yield im provem ent could aim at increasing the harvest index by reducing the hull proportion o f grain and increasing the grain w eight.

In m ost cu ltiv ated rice, the hull proportion varies from 16% to 35% and dry grain w eight from 15 to 40 m g. C an th e hull p roportion o f grain be reduced to less than 10%

and dry grain w eight increased to m ore than 50 m g? T he size and w eight o f a rice grain can be increased eith er by ( !) breeding rice v arieties w ith a larger palea and lem m a (w hich in turn w ould enclose m ore space for the caryopsis to develop) o r by (2) p roducing varieties that respond to P G R application by rapidly increasing the size o f the p alea and lem m a. S uch v arieties can be developed either through conventional breeding o r by the application o f m olecular techniques.

116 Ebenezeretal

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Notes

A uthors' addresses: G .A .I. E benezer, P. D a y an a n d a n , D ep artm en t o f Botany, M adras C hristian C o lleg e, T am b aram 600 0 5 9 , India; S. K rish n an , D e p a rtm en t o f Botany, G o? U niver­

sity, G o a 403 206, India.

Citation: P en g S, H ard y B , ed ito rs. 2001. R ice research fo r food secu rity and poverty allevia­

tion. P roceedings o f the International R ice R esearch C o n feren ce. 3 1 M arch-3 A pril 2.000, L os B anos, P h ilip p in es. L os B anos (P h ilip p in es): International R ice R esearch Institute.

692 p.

Structure of rice caryopsis in relation to strategies for enhancing yield 11.7