Cell cycle events and expression of cell cycle regulators are determining factors in differential grain filling in rice spikelets based on their spatial location on compact panicles.

Rice being a staple crop for human, its production is required to be increased significantly, particularly keeping in view the expected world's population of 9.6 billion by the year 2050. In this context, although the rice breeding programs have been successful in increasing the number of spikelets per panicle, the basal spikelets remain poorly filled, undermining the yield potential. The present study also found the grain filling to bear negative correlation with the panicle grain density. The poorly filled basal spikelets of the compact-panicle cultivars showed a lower endosperm cell division rate and ploidy status of the endosperm nuclei coupled with no significant greater expression of CYCB;1 and CYCH;1 compared with the apical spikelets, unlike that observed in the lax-panicle cultivars, which might have prevented them from overcoming apical dominance. Significantly greater expression of CYCB2;2 in the basal spikelets than in the apical spikelets might also have prevented the former to enter into endoreduplication. Furthermore, expression studies of KRPs in the caryopses revealed that a higher expression of KRP;1 and KRP;4 in the basal spikelets than in the apical spikelets of the compact-panicle cultivars could also be detrimental to grain filling in the former, as KRPs form complex primarily with CDKA-CYCD that promotes S-phase activity and G1/S transition, and thus inhibits endosperm cell division. The study indicates that targeted manipulation of expression of CYCB1;1, CYCB2;2, CYCH1;1, KRP;1 and KRP4 in the basal spikelets of the compact-panicle cultivars may significantly improve their yield performance.

Biochemical and molecular characterisations of salt tolerance components in rice varieties tolerant and sensitive to NaCl: the relevance of Na+ exclusion in salt tolerance in the species.

Soil salinisation is a major abiotic stress in agriculture, and is especially a concern for rice production because among cereal crops, rice is the most salt-sensitive. However, the production of rice must be increased substantially by the year 2050 to meet the demand of the ever growing population. Hence, understanding the biochemical events determining salt tolerance in rice is highly desirable so that the trait can be introduced in cultivars of interest through biotechnological intervention. In this context, an initial study on NaCl response in four Indica rice varieties showed a lower uptake of Na+ in the salt-tolerant Nona Bokra and Pokkali than in the salt-sensitive IR64 and IR29, indicating Na+ exclusion as a primary requirement of salt tolerance in the species. This was also supported by the following features in the salt-tolerant, but not in the -sensitive varieties: (1) highly significant NaCl-induced increase in the activity of PM-H+ATPase, (2) a high constitutive level and NaCl-induced threonine phosphorylation of PM-H+ATPase, necessary to promote its activity, (3) a high constitutive expression of 14-3-3 protein that makes PM-H+ATPase active by binding with the phosphorylated threonine at the C-terminal end, (4) a high constitutive and NaCl-induced expression of SOS1 in roots, and (5) significant NaCl-induced expression of OsCIPK 24, a SOS2 that phosphorylates SOS1. The vacuolar sequestration of Na+ in seedlings was not reflected from the expression pattern of NHX1/NHX1 in response to NaCl. NaCl-induced downregulation of expression of HKTs in roots of Nona Bokra, but upregulation in Pokkali also indicates that their role in salt tolerance in rice could be cultivar specific. The study indicates that consideration of increasing exclusion of Na+ by enhancing the efficiency of SOS1/PM-H+ATPase Na+ exclusion module could be an important aspect in attempting to increase salt tolerance in the rice varieties or cultivars of interest.

Comparative transcriptome profiling of low light tolerant and sensitive rice varieties induced by low light stress at active tillering stage.

Low light intensity is a great limitation for grain yield and quality in rice. However, yield is not significantly reduced in low light tolerant rice varieties. The work therefore planned for comparative transcriptome profiling under low light stress to decipher the genes involved and molecular mechanism of low light tolerance in rice. At active tillering stage, 50% low light exposure for 1 day, 3 days and 5 days were given to Swarnaprabha (low light tolerant) and IR8 (low light sensitive) rice varieties. Illumina (HiSeq) platform was used for transcriptome sequencing. A total of 6,652 and 12,042 genes were differentially expressed due to low light intensity in Swarnaprabha and IR8, respectively as compared to control. CAB, LRP, SBPase, MT15, TF PCL1 and Photosystem I & II complex related gene expressions were mostly increased in Swarnaprabha upon longer duration of low light exposure which was not found in IR8 as compared to control. Their expressions were validated by qRT-PCR. Overall study suggested that the maintenance of grain yield in the tolerant variety under low light might be results of accelerated expression of the genes which enable the plant to keep the photosynthetic processes moving at the same pace even under low light.

Grain density and its impact on grain filling characteristic of rice: mechanistic testing of the concept in genetically related cultivars.

Physiological factors controlling assimilate partitioning was compared in relation to panicle architecture of lax- (Upahar) and compact-panicle (Mahalaxmi) rice cultivars. Grain number and ethylene production at anthesis are low, but filling rate is high in the former compared to high grain number and ethylene production and poor filling trait of the latter. Similar to Mahalaxmi, its progenitors Pankaj and Mahshuri, had attributes of high grain number and grain density, but grain filling was higher and ethylene evolution was lower. Disturbed genetic coherence owing to imbalance of gene groups brought in the cross combinations of Mahshuri and Pankaj could be responsible for high ethylene production leading to semi sterility of Mahalaxmi as the hormone slackened endosperm starch bio-synthesis enzyme activities. Mahalaxmi inherited grain compactness trait of its progenitors, but not the physiological attribute for reduced ethylene production, which impacted grain filling. Upahar, the progeny genotype of Mahalaxmi and IR62 cross, inherited the dominant allele for low ethylene production and good grain filling traits from the high yielding IR62. In conclusion grain filling in compact-panicle rice becomes poor subject to expression of recessive allele for high ethylene production, but the allele is amenable for suppression by corresponding dominant allele in a genetic breeding.

Comparative proteomics of the superior and inferior spikelets at the early grain filling stage in rice cultivars contrast for panicle compactness and ethylene evolution.

The breeding programmes in rice aimed at increasing the number of spikelets per panicle have been accompanied by poor grain filling in the inferior spikelets of large panicle rice, leading to yield disadvantage. The present study attempted to understand the reason for differential grain filling in the inferior and superior spikelets by comparative proteomics considering a compact-panicle rice cultivar Mahalaxmi and a lax-panicle rice cultivar Upahar, which show poor and good grain filling, respectively. An initial study of two rice cultivars for panicle compactness and grain filling revealed an inverse correlation between the two parameters. It was further observed that the panicle compactness in Mahalaxmi was associated with a higher evolution of ethylene by the spikelets, both superior and inferior, compared with the lax-panicle Upahar. The proteomic studies revealed that the superior and inferior spikelets of Mahalaxmi differentially expressed 21 proteins that were also expressed in Upahar. However, in Upahar, only two of these proteins were differentially expressed between the superior and inferior spikelets, indicating that the metabolic activities of the spikelets occupying the superior and inferior positions on the panicle were very different in Mahalaxmi compared with those in Upahar. Among the proteins that were downregulated in the inferior spikelets compared with the superior ones in Mahalaxmi were importin-α, elongation factor 1-ß and cell division control protein 48, which are essential for cell cycle progression and cell division. Low expression of these proteins might inhibit endosperm cell division in the inferior spikelets, limiting their sink capacity and leading to poor grain filling compared to that in the superior spikelets. The poor grain filling in Mahalaxmi may also be a result of the high evolution of ethylene in the inferior spikelets, which is reflected from the observation that these spikelets showed significantly higher expression of S-adenosylmethionine synthase and the gene encoding the enzyme than the superior spikelets in this cultivar, but not in Upahar; S-adenosynlmethionine synthase catalyses the synthesis of S-adenosylmethionine, the precursor of ethylene biosynthesis.

Biochemical and molecular characterisation of salt-induced poor grain filling in a rice cultivar.

Despite the prevalence of poor grain filling in rice (Oryza sativa L.) under abiotic stress, the reason for this is largely unexplored. Application of 0.75% NaCl to a salt-sensitive rice cultivar at late booting resulted in a >20% yield loss. Spikelets per panicle and the percentage of filled grain decreased significantly in response to NaCl application. The inhibitory effect of NaCl on grain filling was greater in basal than in apical spikelets. Sucrose synthase (SUS) activity was positively correlated with grain weight. The transcript levels of the SUS isoforms differed greatly: the levels of SUS2 increased significantly in response to salt; those of SUS4 decreased drastically. Gene expression studies of starch synthase and ADP-glucose pyrophosphorylase showed that the decreased transcript levels of one isoform was compensated by an increase in those of the other. Salt application also significantly increased the gene expression of the ethylene receptors and the ethylene signalling proteins. The increase in their transcript levels was comparatively greater in basal than in apical spikelets. Significant enhancement in the transcript levels of the ethylene receptors and the increase in the production of ethylene indicated that the salt-induced inhibition of grain filling might be mediated by ethylene. Additionally, the inhibition of chromosomal endoreduplication mediated by decreased transcript levels of B-type cyclin could explain poor grain filling under salt stress. A significant increase in the transcript levels of the ethylene-responsive factors in the spikelets during grain filling in response to salt indicated their possible protective role in grain filling under stress.

Spikelet-specific variation in ethylene production and constitutive expression of ethylene receptors and signal transducers during grain filling of compact- and lax-panicle rice (Oryza sativa) cultivars.

Grain yields in modern super rice cultivars do not always meet the expectations because many spikelets are located on secondary branches in closely packed homogeneous distribution in these plants, and they do not fill properly. The factors limiting grain filling of such spikelets, especially in the lower panicle branches, are elusive. Two long-duration rice cultivars differing in panicle density, Mahalaxmi (compact) and Upahar (lax), were cultivated in an open field plot. Grain filling, ethylene production and constitutive expression of ethylene receptors and ethylene signal transducers in apical and basal spikelets of the panicle were compared during the early post-anthesis stage, which is the most critical period for grain development. In another experiment, a similar assessment was made for the medium-duration cultivars compact-panicle OR-1918 and lax-panicle Lalat. Grain weight of the apical spikelets was always higher than that of the basal spikelets. This gradient of grain weight was wide in the compact-panicle cultivars and narrow in the lax-panicle cultivars. Compared to apical spikelets, the basal spikelets produced more ethylene at anthesis and retained the capacity for post-anthesis expression of ethylene receptors and ethylene signal transducers longer. High ethylene production enhanced the expression of the RSR1 gene, but reduced expression of the GBSS1 gene. Ethylene inhibited the partitioning of assimilates of developing grains resulting in low starch biosynthesis and high accumulation of soluble carbohydrates. It is concluded that an increase in grain/spikelet density in rice panicles reduces apical dominance to the detriment of grain filling by production of ethylene and/or enhanced perception of the ethylene signal. Ethylene could be a second messenger for apical dominance in grain filling. The manipulation of the ethylene signal would possibly improve rice grain yield.

Isolation, identification and expression analysis of salt-induced genes in Suaeda maritima, a natural halophyte, using PCR-based suppression subtractive hybridization.

BACKGROUND: Despite wealth of information generated on salt tolerance mechanism, its basics still remain elusive. Thus, there is a need of continued effort to understand the salt tolerance mechanism using suitable biotechnological techniques and test plants (species) to enable development of salt tolerant cultivars of interest. Therefore, the present study was undertaken to generate information on salt stress responsive genes in a natural halophyte, Suaeda maritima, using PCR-based suppression subtractive hybridization (PCR-SSH) technique. RESULTS: Forward and reverse SSH cDNA libraries were constructed after exposing the young plants to 425 mM NaCl for 24 h. From the forward SSH cDNA library, 429 high quality ESTs were obtained. BLASTX search and TIGR assembler programme revealed overexpression of 167 unigenes comprising 89 singletons and 78 contigs with ESTs redundancy of 81.8%. Among the unigenes, 32.5% were found to be of special interest, indicating novel function of these genes with regard to salt tolerance. Literature search for the known unigenes revealed that only 17 of them were salt-inducible. A comparative analysis of the existing SSH cDNA libraries for NaCl stress in plants showed that only a few overexpressing unigenes were common in them. Moreover, the present study also showed increased expression of phosphoethanolamine N-methyltransferase gene, indicating the possible accumulation of a much studied osmoticum, glycinebetaine, in halophyte under salt stress. Functional categorization of the proteins as per the Munich database in general revealed that salt tolerance could be largely determined by the proteins involved in transcription, signal transduction, protein activity regulation and cell differentiation and organogenesis. CONCLUSION: The study provided a clear indication of possible vital role of glycinebetaine in the salt tolerance process in S. maritima. However, the salt-induced expression of a large number of genes involved in a wide range of cellular functions was indicative of highly complex nature of the process as such. Most of the salt inducible genes, nonetheless, appeared to be species-specific. In light of the observations made, it is reasonable to emphasize that a comparative analysis of ESTs from SSH cDNA libraries generated systematically for a few halophytes with varying salt exposure time may clearly identify the key salt tolerance determinant genes to a minimum number, highly desirable for any genetic manipulation adventure.