Post-anthesis development of inferior and superior spikelets in rice in relation to abscisic acid and ethylene

The purpose of this study was to test the hypothesis that the interaction between abscisic acid (ABA) and ethylene may be involved in mediating the post-anthesis development of spikelets in rice (Oryza sativa L.). Two rice genotypes were field-grown, and the changes of ABA, ethylene, and 1-aminocylopropane-1-carboxylic acid (ACC) levels in spikelets during grain filling and their relationships with endosperm-division and grain-filling rates were investigated. The results showed that earlier-flowering superior spikelets exerted dominance over later-flowering inferior spikelets in endosperm cell-division and grain-filling rates. The two genotypes behaved the same. Later-flowering spikelets had higher levels of ethylene and ACC than earlier-flowering spikelets. The ethylene evolution rate was significantly and negatively correlated with the cell division and grain filling rates. By contrast to ethylene, later-flowering spikelets contained a lower ABA content/concentration and showed a low content ratio of ABA to ACC than earlier-flowering ones. The cell-division and grain-filling rates were significantly and positively correlated with both ABA contents and the ratio of ABA to ACC. Application of cobalt ion (inhibitor of ethylene synthesis) or ABA at an early grain-filling stage significantly increased endosperm cell division rate and cell number, grain-filling rate, and grain weight of inferior spikelets. Application of ethephon (an ethylene-releasing agent) or fluridone (an inhibitor of carotenoid synthesis) had the opposite effect. The results suggest that antagonistic interactions between ABA and ethylene mediate endosperm cell-division and grain-filling in rice. A higher ratio of ABA to ethylene in rice spikelets is required to maintain a faster grain-filling rate.     

Polyamine and ethylene interactions in grain filling of superior and inferior spikelets of rice

This study was to test the hypothesis that polyamines (PAs) and ethylene and their interactions may be involved in mediating the post-anthesis development of spikelets in rice (Oryza sativa L.). Six rice cultivars differing in grain filling rate were field-grown, and the changing patterns of PAs and ethylene levels in rice spikelets during the filling and their relations with grain filling rates were investigated. The results showed that inferior spikelets had much greater ethylene evolution rate and 1-aminocylopropane-1-carboxylic acid (ACC) concentration than superior spikelets. Opposite to ethylene production, superior spikelets showed much higher free-spermidine (Spd) and free-spermine (Spm) concentrations than inferior spikelets. Grain filling rate was very significantly and negatively correlated with ethylene evolution rate and ACC concentration, whereas positively correlated with free-Spd and free-Spm concentrations and with the ratio of free-Spd or free-Spm to ACC. Application of Spd, Spm, or aminoethoxyvinylglycine (an inhibitor of ethylene synthesis by inhibiting ACC synthesis) to panicles at the early grain filling stage significantly reduced ethylene evolution rate and ACC concentration, while significantly increased Spd and Spm concentrations, grain filling rate and grain weight of inferior spikelets. Application of ACC, ethephon (an ethylene-releasing agent), or methylglyoxal-bis (guanylhydrazone) (an inhibitor of Spd and Spm synthesis) showed the opposite effects. The results suggest that antagonistic interactions between PAs (Spd and Spm) and ethylene may be involved in mediating grain filling. A higher ratio of free-Spd or free-Spm to ethylene in rice spikelets could enhance grain filling.     

Involvement of polyamines in the post-anthesis development of inferior and superior spikelets in rice

Early-flowered superior spikelets usually exhibit a faster grain filling rate and heavier grain weight than late-flowered inferior spikelets in rice (Oryza sativa L.). But the intrinsic factors responsible for the variations between the two types of spikelets are unclear. This study investigated whether and how polyamines (PAs) are involved in regulating post-anthesis development of rice spikelets. Six rice genotypes differing in grain filling rate were field grown, and PA levels and activities of the enzymes involved in PA biosynthesis were measured in both superior and inferior spikelets. The results showed that superior spikelets exhibited higher levels of free spermidine (Spd) and free spermine (Spm) and higher activities of arginine decarboxylase (ADC, EC 4.1.1.19), S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) and Spd synthase (EC 2.5.1.16) than inferior spikelets at the early endosperm cell division and grain filling stage. The maximum concentrations of free Spd and free Spm and the maximum activities of ADC, SAMDC and Spd synthase were significantly correlated with the maximum cell division and grain filling rates, maximum cell number and grain weight. Application of Spd and Spm to panicles resulted in significantly higher rates of endosperm cell division and grain filling in inferior spikelets along with the activities of sucrose synthase (EC 2.4.1.13), ADP glucose pyrophosphorylase (EC 2.7.7.27) and soluble starch synthase (EC 2.4.1.21), suggesting that these PAs are involved in the sucrose-starch metabolic pathway. The results indicate that the poor development of inferior spikelets is attributed, at least partly, to the low PA level and its low biosynthetic activity.     

Hormones in the grains and roots in relation to post-anthesis development of inferior and superior spikelets in japonica/indica hybrid rice

Grain filling is usually not adequate in later-flowering inferior spikelets in japonica/indica (J/I) hybrid rice (Oryza sativa) although it shows stronger hybrid vigor than indica/indica (I/I) hybrid. This study investigated the potential causes by examining changes in zeatin (Z) + zeatin riboside (ZR), indole-3-acetic acid (IAA), gibberellins (GAs, GA₁ + GA₄), and abscisic acid (ABA) in spikelets and roots during the grain filling period. The inferior spikelets of J/I hybrid exhibited low rate of endosperm cell division and slow grain filling. During the early grain filing period, they had less Z + ZR, IAA, and ABA, but more GAs, than the earlier-flowering superior spikelets. If compared to the inferior spikelets of the I/I hybrid, the J/I inferior spikelets also had less Z + ZR, IAA, and ABA. Rates of endosperm cell division and grain filling were positively and significantly correlated with Z + ZR and ABA contents in both grains and roots or IAA in grains, whereas not significantly correlated with GAs either in grains or roots or IAA in roots. Applications of kinetin, IAA, or ABA to spikelets, or kinetin and ABA to roots, enhanced cell division and grain filling in the inferior spikelets. Results suggest that low contents of cytokinins and ABA in both grains and roots and low contents of IAA in grains may result in the poor filling of inferior spikelets in the J/I hybrid.     

Hormones in the grains in relation to sink strength and postanthesis development of spikelets in rice

Inferior spikelets usually exhibit a slower grain filling rate and lower grain weight than superior spikelets in a rice (Oryza sativa L.) panicle. This study investigated whether the variations in grain filling between the two kinds of spikelets were attributed to their sink strength and whether the sink strength was regulated by the hormonal levels in the grains. Using two field-grown rice genotypes, the division rate of endosperm cells, hormonal levels in the grains, and grain weight of both superior and inferior spikelets were determined during the grain filling period. The results showed that superior spikelets had dominance over inferior spikelets in endosperm cell division rate and cell number, grain filling and grain weight. Changes in zeatin (Z) and zeatin riboside (ZR) contents paralleled and were very significantly correlated with the cell division rate and cell number. Cell division rate and the content of indole-3-acetic acid (IAA) in the grains were also significantly correlated. Gibberellin (GAs; GA₁+ GA₄) content of the grains was high but ABA levels were low at the early grain filling stage. ABA increased substantially during the linear phase of grain growth and was very significantly correlated with grain dry weight during this period. Application of kinetin at 2 through 6 days post anthesis (DPA) significantly increased cell number, while spraying ABA at 11 through 15 DPA significantly increased the grain filling rate. The results suggest that differences in sink strength are responsible for variations in grain filling between superior and inferior spikelets. Both cytokinins and IAA in the grains may mediate cell division in rice endosperm at early grain filling stages, and therefore regulate the sink size of the grain, whereas ABA content correlates with sink activity during the linear period of grain growth.     

Growth characteristics and endosperm structure of superior and inferior spikelets of indica rice under high-temperature stress

Heat stress severely reduces rice yield and quality; however, differences between the superior, early-flowering and inferior, later-flowering spikelets of indica rice in response to high-temperature stress during grain filling remain unclear. This study investigated the effects of high temperature (HT, 33.6/20.7 °C day/night) on growth, endosperm structure, and hormone and polyamine content of superior and inferior spikelets of heat-sensitive (SG-1) and heat-tolerant (HHZ) indica cultivars. The HT decreased fertilization rate, caused earlier grain filling, and reduced duration of grain filling, thus resulting in decreased grain mass and a poor endosperm structure. In addition, soluble sugar and sucrose content increased, and starch synthesis decreased by HT at the early stage of grain filling. The HT increased polyamine [spermidine (Spd) and spermine (Spm)] and abscisic acid (ABA) content, but reduced zeatin (Z) + zeatin riboside (ZR) and indole-3-acetic acid (IAA) content in the grains. Such effects were more apparent in the inferior than superior spikelets; however, the inferior spikelets of SG-1 were more affected than those of HHZ. At the middle grain filling stage, HT produced little difference between the two cultivars. Our results suggest that the poor development of inferior spikelets of SG-1 under the HT could be attributed, at least in part, to the changed content and ratios of free polyamines [putrescine (Put), Spd, and Spm] and phytohormones (Z+ZR, IAA, and ABA) and the conversion efficiency of sucrose into starch.     

Glutelin accumulation and changes in the levels of its mRNA in the superior and inferior spikelets of rice ear during ripening

Glutelin accumulation in the apical spikelet of the top primary branch (superior spikelet) and the second spikelet of the lowest secondary branch (inferior spikelet) of the ear of the rice plant (Oryza sativa L.) was characterized during grain filling.In the superior spikelet, the accumulation of dry matter and nitrogen started immediately after flowering and rapidly reached the maturation level by 20 days after heading (DAH). At 7 DAH, total RNA content had already reached its maximum level and glutelin mRNA content 70% of its maximum. The increase in glutelin mRNA was followed by a rapid increase in glutelin between 7 and 16 DAH.In the inferior spikelet dry matter, nitrogen and glutelin accumulation were low immediately after flowering and increased only after grain filling of the superior spikelet was almost complete. Total RNA and glutelin mRNA increased much later at slower rates than in the superior spikelet.It is very likely that the retardation of dry matter, total nitrogen and glutelin accumulation in the inferior spikelet is due to retardation of differentiation and development of endosperm tissue, and to glutelin gene expression in endosperm cells. It is suggested that the delayed development resulted from limited partitioning of nutrients to the inferior spikelet at the early stage of ripening.     

Endosperm enrichment and physicochemical properties of superior and inferior grain starch in super hybrid rice

• A significant asynchronous phenomenon exists in super hybrid rice because of the differences in spike and spikelet positions, which affect the accumulation and properties of starch. However, little is known about the endosperm enrichment and physicochemical properties of starch in superior and inferior grains in super hybrid rice.
• Rice YY2640 was selected as study material to investigate the enrichment and physicochemical properties of starch in superior and inferior grains in super rice using semi‐thin sections, X‐ray diffraction and related technologies.
• Superior grain filling was a continuous process, whereas inferior grain only started 8–10 days after anthesis. The order of starch accumulation starts in the central endosperm, then in the endosperm of the proximal vascular bundle and finally in the aleurone layer. Compared with the inferior grains, the superior grains have a higher 1000‐grain weight, apparent amylose content, total starch content, average starch granule size, relative crystallinity, solubility and a resonance peak ratio at 1022/995 cm^?1, whereas the swelling power and ratio of the resonance peak at 1045/1022 cm^?1 were lower. The final degree of hydrolysis of HCl, AAG and PPA of the superior grains were significantly lower than those of the inferior grains.
• The findings indicate that the different physicochemical properties of starch were mainly related to the development order of superior and inferior grains and the spatial enrichment of starch.

     

Regulation of genes associated with auxin, ethylene and ABA pathways by 2,4-dichlorophenoxyacetic acid in Arabidopsis

The chemical 2,4-dichlorophenoxyacetic acid (2,4-D) regulates plant growth and development and mimics auxins in exhibiting a biphasic mode of action. Although gene regulation in response to the natural auxin indole acetic acid (IAA) has been examined, the molecular mode of action of 2,4-D is poorly understood. Data from biochemical studies, (Grossmann (2000) Mode of action of auxin herbicides: a new ending to a long, drawn out story. Trends Plant Sci 5:506–508) proposed that at high concentrations, auxins and auxinic herbicides induced the plant hormones ethylene and abscisic acid (ABA), leading to inhibited plant growth and senescence. Further, in a recent gene expression study (Raghavan et al. (2005) Effect of herbicidal application of 2,4-dichlorophenoxyacetic acid in Arabidopsis. Funct Integr Genomics 5:4–17), we have confirmed that at high concentrations, 2,4-D induced the expression of the gene NCED1, which encodes 9-cis-epoxycarotenoid dioxygenase, a key regulatory enzyme of ABA biosynthesis. To understand the concentration-dependent mode of action of 2,4-D, we further examined the regulation of whole genome of Arabidopsis in response to a range of 2,4-D concentrations from 0.001 to 1.0 mM, using the ATH1-121501 Arabidopsis whole genome microarray developed by Affymetrix. Results of this study indicated that 2,4-D induced the expression of auxin-response genes (IAA1, IAA13, IAA19) at both auxinic and herbicidal levels of application, whereas the TIR1 and ASK1 genes, which are associated with ubiquitin-mediated auxin signalling, were down-regulated in response to low concentrations of 2,4-D application. It was also observed that in response to low concentrations of 2,4-D, ethylene biosynthesis was induced, as suggested by the up-regulation of genes encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. Although genes involved in ethylene biosynthesis were not regulated in response to 0.1 and 1.0 mM 2,4-D, ethylene signalling was induced as indicated by the down-regulation of CTR1 and ERS, both of which play a key role in the ethylene signalling pathway. In response to 1.0 mM 2,4-D, both ABA biosynthesis and signalling were induced, in contrast to the response to lower concentrations of 2,4-D where ABA biosynthesis was suppressed. We present a comprehensive model indicating a molecular mode of action for 2,4-D in Arabidopsis and the effects of this growth regulator on the auxin, ethylene and abscisic acid pathways. Experiment station: Plant Biotechnology Centre, Primary Industries Research Victoria, Department of Primary Industries, La Trobe University, Bundoora, Victoria 3086, and the Victorian Microarray Technology Consortium (VMTC).     

Activities of key enzymes for starch synthesis in relation to growth of superior and inferior grains on winter wheat (Triticum aestivum L.) spike

Weight of individual grains is a major yield component in wheat. The non-uniform distribution of single grain weight on a wheat spike is assumed to be closely associated with starch synthesis in grains. The present study was undertaken to determine if the enzymes involved in starch synthesis cause the differences in single grain weight between superior and inferior grains on a wheat spike. Using two high-yield winter wheat (Triticum aestivum L.) varieties differing in grain weight and three nitrogen rates for one variety, the contents of amylose and amylopectin, and activities of enzymes involved in starch synthesis in both superior and inferior grains were investigated during the entire period of grain filling. Superior grains showed generally higher starch accumulation rates and activities of enzymes including SS (sucrose synthase), UDPGPPase (UDP-glucose pyrophosphorylase), ADPGPPase (ADP-glucose pyrophosphorylase), SSS (soluble starch synthase) and GBSS (starch granule bound starch synthase) and subsequently produced much higher single grain weight than inferior grains. Nitrogen increased enzyme activities and starch accumulation rates, and thus improved individual grain weight, especially for inferior grains. The SS, ADPGPPase and SSS were significantly correlated to amylopectin accumulation, while SS, ADPGPPase, SSS and GBSS were significantly correlated to amylose accumulation. This infers that SS, ADPGPPase and starch synthase play key roles in regulating starch accumulation and grain weight in superior and inferior grains on a wheat spike.     

Comparison of the starch synthesis genes between maize and rice: copies, chromosome location and expression divergence

Gene duplication and divergence are important evolutionary processes. It has been suggested that a whole genome duplication (WGD) event occurred in the Gramineae, predating its divergence, and a second WGD occurred in maize during its evolution. In this study we compared the fate of the genes involved in the core pathway of starch biosynthesis following the ancient and second WGDs in maize and rice. In total, thirty starch synthesis genes were detected in the maize genome, which covered all the starch synthesis gene families encoded by 27 genes in rice. All of these genes, except ZmGBSSIIb and ZmBEIII, are anchored within large-scale synteny blocks of rice and maize chromosomes. Previous findings and our results indicate that two of the current copies of many starch synthesis genes (including AGPL, AGPS, GBSS, SSII, SSIII, and BEII) probably arose from the ancient WGD in the Gramineae and are still present in the maize and rice genome. Furthermore, two copies of at least six genes (AGPS1, SSIIb, SSIIIb, GBSSII, BEI, and ISA3) appear to have been retained in the maize genome after its second WGD, although complete coding regions were only detected among the duplicate sets of AGPS1, SSIIb, and SSIIIb. The expression patterns of the remaining duplicate sets of starch synthesis genes (AGPL1/2, AGPS1/2, SSIIa/b, SSIIIa/b, GBSSI/II, and BEIIa/b) differ in their expression and could be classified into two groups in maize. The first group is mainly expressed in the endosperm, whereas the second is expressed in other organs and the early endosperm development. The four duplicate sets of ZmGBSSII, ZmSSIIb, ZmSSIIIb and AGPS1, which arose from the second WGD diverged in gene structure and/or expression patterns in maize. These results indicated that some duplicated starch synthesis genes were remained, whereas others diverged in gene structure and/or expression pattern in maize. For most of the duplicated genes, one of the copies has disappeared in the maize genome after the WGD and the subsequent “diploidization”. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mutations of genes in synthesis of the carotenoid precursors of ABA lead to pre-harvest sprouting and photo-oxidation in rice

Pre-harvest sprouting (PHS) or vivipary in cereals is an important agronomic trait that results in significant economic loss. A considerable number of mutations that cause PHS have been identified in several species. However, relatively few viviparous mutants in rice ( Oryza sativa L.) have been reported. To explore the mechanism of PHS in rice, we carried out an extensive genetic screening and identified 12 PHS mutants ( phs ). Based on their phenotypes, these phs mutants were classified into three groups. Here we characterize in detail one of these groups, which contains mutations in genes encoding major enzymes of the carotenoid biosynthesis pathway, including phytoene desaturase (OsPDS), ζ-carotene desaturase (OsZDS), carotenoid isomerase (OsCRTISO) and lycopene β -cyclase (β-OsLCY), which are essential for the biosynthesis of carotenoid precursors of ABA. As expected, the amount of ABA was reduced in all four phs mutants compared with that in the wild type. Chlorophyll fluorescence analysis revealed the occurrence of photoinhibition in the photosystem and decreased capacity for eliminating excess energy by thermal dissipation. The greatly increased activities of reactive oxygen species (ROS) scavenging enzymes, and reduced photosystem (PS) II core proteins CP43, CP47 and D1 in leaves of the Oscrtiso / phs3-1 mutant and OsLCY RNAi transgenic rice indicated that photo-oxidative damage occurred in PS II, consistent with the accumulation of ROS in these plants. These results suggest that the impairment of carotenoid biosynthesis causes photo-oxidation and ABA-deficiency phenotypes, of which the latter is a major factor controlling the PHS trait in rice.     

Structure of rice starch and its relation to cooked-rice texture

Starch from seven varieties of rice, known to cook from very soft to very hard texture, was fractionated by gel-permeation chromatography on Sepharose CL-2B column. The high-molecular-weight fraction (Sepharose FRI) and the low-molecular-weight fraction (Sepharose FRII, further sub-divided into FRIIa, IIb and IIc) were debranched using isoamylase and fractionated on Biogel P-10. All the four fractions in all the different varieties of rice gave a similar trimodal chain profile, indicating the presence of branched molecules in all of them. Clearly, the branched component of starch (‘amylopectin’) is not necessarily big in size, but includes very small to very big molecules. The presence or absence of the largely linear, and relatively small molecule, ‘amylose’, could not be settled either way with the technique employed. However, based on certain assumptions, amylose content was calculated to be in the range of 7%–11% in the samples, much less than generally thought. The content of long-B chains of the branched molecule in the four Sepharose fractions individually and in aggregate, as well as the calculated amylose content, correlated well with the sensory tenderness of cooked rice. It was observed that the content of all long linear chains, including amylose if any, govern the texture of cooked rice.     

Regulation of gene expression by endogenous ABA in tomato plants

In response to water deficit, endogenous abscisic acid (ABA) accumulates in plants. This ABA serves as a signal for a multitude of processes, including regulation of gene expression. ABA accumulated in response to water deficit signals cellular as well as whole plant responses playing a role in the pattern of gene expression throughout the plant. Although the function of genes regulated by ABA during stress are currently poorly understood, a number of these genes may permit the plant to adapt to environmental stress.     

Microsatellites in starch-synthesizing genes in relation to starch physicochemical properties in waxy rice ( Oryza sativa L.)

Rice starch is composed of amylose and amylopectin. Amylose content, an important determinant of rice starch quality, is primarily controlled by the waxy gene, encoding granule-bound starch synthase (GBSS). The starch branching enzyme (SBE) and soluble starch synthase (SSS) play major roles in the synthesis of amylopectin. Microsatellite polymorphisms in the three genes, the wx gene encoding granule-bound starch synthase I, the SBE gene encoding starch branching enzyme I and the SSS gene encoding soluble starch synthase I, were studied for 56 accessions of waxy rice ( Oryza sativa L.). Four (CT)(n) microsatellite alleles, (CT)(16), (CT)(17), (CT)(18) and (CT)(19), at the wx locus were detected in this set of waxy rice, of which (CT)(17) was the most frequent. Three (CT)(n) microsatellite allele classes were found at the SBE locus, (CT)(8) or (CT)(10) together with an insertion sequence of CTCTCGGGCGA, and (CT)(8) alone without the insertion. There were multiple microsatellites clustered at the SSS locus. However, these alleles can also be grouped into three classes, i.e. the allele class SSS-A = (AC)(2) em leader TCC(TC)(11) em leader (TC)(5)C(ACC)(11), the allele class SSS-B = (AC)(3) em leader TCT(TC)(6) em leader (TC)(4)C(ACC)(9), and the allele class SSS-C = (AC)(3) em leader TCT(TC)(6) em leader (TC)(4)C(ACC)(8). The analyses of starch physicochemical properties among different microsatellite genotypes indicated that the waxy rice group with the (CT)(19) allele, the SBE-A allele and the SSS-B allele was quite different from other groups. Nine out of 15 accessions with a high gelatinization temperature (GT) belonged to the wx (CT)(19) group, all of them belonged to the SBE-A group and 13 of them belonged to the SSS-B group. These microsatellites might be useful in marker-assisted breeding for the improvement of rice grain quality.