Analysis of seed production and seed shattering in a new artificial grassland forage: pigeon pea.

Pigeon pea is a perennial leguminous plant that is widely cultivated as a forage and pharmaceutical plant in subtropical and tropical areas, especially in artificial grasslands. Higher seed shattering is one of the most important factors in potentially increasing the seed yield of pigeon pea. Advance technology is necessary to increase the seed yield of pigeon pea. Through 2 consecutive years of field observations, we found that fertile tiller number was the key component of the seed yield of pigeon pea due to the direct effect of fertile tiller number per plant (0.364) on pigeon pea seed yield was the highest. Multiplex morphology, histology, and cytological and hydrolytic enzyme activity analysis showed that shatter-susceptible and shatter-resistant pigeon peas possessed an abscission layer at the same time (10 DAF); however, abscission layer cells dissolved earlier in shattering-susceptible pigeon pea (15 DAF), which led to the tearing of the abscission layer. The number of vascular bundle cells and vascular bundle area were the most significant negative factors (p< 0.01) affecting seed shattering. Cellulase and polygalacturonase were involved in the dehiscence process. In addition, we inferred that larger vascular bundle tissues and cells in the ventral suture of seed pods could effectively resist the dehiscence pressure of the abscission layer. This study provides foundation for further molecular studies to increase the seed yield of pigeon pea.

Histological characteristics, cell wall hydrolytic enzyme activity, and transcriptome analysis with seed shattering of Stylosanthes accessions.

Stylosanthes spp. (stylo) are annual or perennial legume forages that are widely grown as forage and cover crops in tropical and subtropical regions. However, the seed yield of stylo is very low due to serious seed shattering. In the present study, we found that, although seed shattering was common among the stylo accessions, the shattering rates were genetically different. Therefore, we first synthesized the morphological, histological characteristic, physiochemical, and transcriptome analyses to determine the seed shattering mechanism in stylo. In general, the stylo germplasm with shorter lobules and thicker stems had a lower seed shattering rate and a higher seed weight. The seed and seed stalk joint is the abscission zone in stylo. Multiplex histology and hydrolytic enzyme activity analysis showed that the tearing of the abscission zone occurs due to the intense enzymatic degradation of polygalacturonase and cellulase in the seed shattering-susceptible accession TF0275. cDNA libraries from the abscission zone tissues of TF0041 and TF0275 at 14, 21, and 28 days after flowering were constructed and sequenced. A total of 47,606 unigenes were annotated and 18,606 differentially expressed genes (DEGs) were detected, including 9,140 upregulated and 9,446 downregulated unigenes. Furthermore, the 26 candidate DEGs involved in lignin biosynthesis, cellulase synthesis, and plant hormone signal transduction were found at all three developmental stages. This study provides valuable insights for future mechanistic studies of seed shattering in stylo.

Integrated multi-omics analysis provides insights into genome evolution and phosphorus deficiency adaptation in pigeonpea (Cajanus cajan).

Pigeonpea (Cajanus cajan) is an important legume food crop and plays a crucial role in a secure food supply in many developing countries. Several previous studies have suggested that pigeonpea has great potential for phosphorus (P) deficiency tolerance, but little is known about the underlying mechanism. In this study, the physiological and molecular responses of pigeonpea roots to phosphate (Pi) starvation were investigated through integrating phenotypic, genomic, transcriptomic, metabolomic, and lipidomic analyses. The results showed that low-Pi treatment increased total root length, root surface area, and root acid phosphatase activity, and promoted the secretion of organic acids (e.g. citric acids, piscidic acids, and protocatechuic acids) and the degradation of phospholipids and other P-containing metabolites in the roots of pigeonpea. Consistent with the morphological, physiological, and biochemical changes, a large number of genes involved in these Pi-starvation responses were significantly upregulated in Pi-deficient pigeonpea roots. Among these Pi-starvation response genes upregulated by low-Pi treatment, four gene families were expanded through recent tandem duplication in the pigeonpea genome, namely phosphate transporter 1 (PHT1), phosphoethanolamine/phosphocholine phosphatase (PECP), fasciclin-like arabinogalactan protein (FLA), and glutamate decarboxylase (GAD). These gene families may be associated with Pi uptake from the soil, phospholipid recycling, root morphological remodeling, and regulation of organic acid exudation. Taken together, our results suggest that pigeonpea employs complex Pi-starvation responses to strengthen P acquisition and utilization during low-Pi stress. This study provides new insights into the genome evolution and P deficiency adaptation mechanism of pigeonpea.

A root-associated purple acid phosphatase, SgPAP23, mediates extracellular phytate-P utilization in Stylosanthes guianensis.

As a major component of soil organic phosphorus (P), phytate-P is unavailable to plants unless hydrolysed by phytase to release inorganic phosphate. However, knowledge on natural variation in root-associated phytase activity and its underlying molecular mechanisms in plants remains fragmentary. In this study, variations in root internal and associated phytase activity were observed among 39 genotypes of Stylosanthes guianensis (Stylo), which is well adapted to acid soils. Furthermore, TPRC2001-1, the genotype with the highest root-associated phytase activity, was more capable of utilizing extracellular phytate-P than Fine-stem, the genotype with the lowest root-associated phytase activity. After protein liquid chromatography-tandem mass spectrometry analysis, a purple acid phosphatase (PAP), SgPAP23, was identified and cloned from TPRC2001-1. SgPAP23 exhibited high activity against phytate-P and was mainly localized on the plasma membrane. Furthermore, SgPAP23 overexpression resulted in significant increases of root-associated phytase activity and thus facilitated extracellular phytate-P utilization in both bean (Phaseolus vulgaris) hairy roots and Arabidopsis thaliana. The results herein support the conclusion that SgPAP23 is a primary contributor to the superior extracellular phytate-P utilization in stylo and thus is used to develop cultivars with efficient extracellular phytate-P utilization.

Genetic control of the root system in rice under normal and drought stress conditions by genome-wide association study.

A variety of adverse conditions including drought stress severely affect rice production. Root system plays a critical role in drought avoidance, which is one of the major mechanisms of drought resistance. In this study, we adopted genome-wide association study (GWAS) to dissect the genetic basis controlling various root traits by using a natural population consisting of 529 representative rice accessions. A total of 413 suggestive associations, containing 143 significant associations, were identified for 21 root traits, such as maximum root length, root volume, and root dry weight under normal and drought stress conditions at the maturation stage. More than 80 percent of the suggestive loci were located in the region of reported QTLs for root traits, while about 20 percent of suggestive loci were novel loci detected in this study. Besides, 11 reported root-related genes, including DRO1, WOX11, and OsPID, were found to co-locate with the association loci. We further proved that the association results can facilitate the efficient identification of causal genes for root traits by the two case studies of Nal1 and OsJAZ1. These loci and their candidate causal genes provide an important basis for the genetic improvement of root traits and drought resistance.

Characterization of a novel cyclase-like gene family involved in controlling stress tolerance in rice.

A novel cyclase-like gene family (CYL) encodes proteins containing cyclase domain, but their functions are largely unknown. We report the systematic identification and characterization of CYL genes in the rice genome. Five putative CYL protein sequences (OsCYL1 to 4b) were identified. These sequences and other CYL homologs were classified into four subgroups based on phylogenetic analysis. Distinct diversification of these CYL proteins exists between plants and non-plants. The CYL family has conserved exon-intron structures, and the organizations of putative motifs in plants are specifically diverse. All OsCYL genes were expressed in a wide range of tissues or organs and were responsive to at least one of the abiotic stresses and hormone treatments applied. Protein OsCYL4a is targeted to the cell membrane. The overexpression of one stress-responsive gene OsCYL4a in rice resulted in decreased tolerance to salt, drought, cold, and oxidative stress. The expression levels of some abiotic stress-responsive factors, including H2O2-accumulating negative factors DST and OsSKIPa in OsCYL4a-overexpressing plants, were reduced compared with the wild type under normal condition and drought stress. These results suggest that rice CYL family may be functionally conserved polyketide cyclase, resulting in the rapid accumulation of reactive oxygen species to decrease tolerance to abiotic stresses.

Insight into differential responses of upland and paddy rice to drought stress by comparative expression profiling analysis.

In this study, the drought responses of two genotypes, IRAT109 and Zhenshan 97 (ZS97), representing upland and paddy rice, respectively, were systematically compared at the morphological, physiological and transcriptional levels. IRAT109 has better performance in traits related to drought avoidance, such as leaf rolling, root volumes, the ratio of leaf water loss and relative conductivity. At the transcriptional level, more genes were induced by drought in IRAT109 at the early drought stage, but more genes had dynamic expression patterns in ZS97 at different drought degrees. Under drought conditions, more genes related to reproductive development and establishment of localization were repressed in IRAT109, but more genes involved in degradation of cellular components were induced in ZS97. By checking the expression patterns of 36 drought-responsive genes (located in 14 quantitative trail loci [QTL] intervals) in ZS97, IRAT109 and near isogenic lines (NILs) of the QTL intervals, we found that more than half of these genes had their expression patterns or expression levels changed in the NILs when compared to that in ZS97 or IRAT109. Our results may provide valuable information for dissecting the genetic bases of traits related to drought resistance, as well as for narrowing the candidate genes for the traits.

Evaluation of near-isogenic lines for drought resistance QTL and fine mapping of a locus affecting flag leaf width, spikelet number, and root volume in rice.

Drought stress is a major limiting factor for crop production and breeding for drought resistance is very challenging due to the complex nature of this trait. Previous studies in rice suggest that the upland japonica variety IRAT109 shows better drought resistance than the lowland indica variety Zhenshan 97. Numerous quantitative trait loci (QTL) have been previously mapped using a recombinant inbred line population derived from these two genotypes. In this study, near-isogenic lines (NILs) for 17 drought resistance-related QTL were constructed and phenotypic variations of these NILs were investigated under drought and normal conditions. Fourteen of these NILs showed significant phenotypic differences relative to the recurrent parent under at least one of the conditions and nine NILs showed significant differences under both conditions. After eliminating the effect of heading date on drought resistance, only four NILs carrying seven QTL (four for the same grain yield-related traits and three for the same or similar root traits QTL) showed differences consistent with the original QTL mapping results. One of these lines (N19) contains qFSR4, a QTL on chromosome 4 controlling root volume per tiller and co-segregating with flag leaf width and spikelet number per panicle. Using a population derived from N19, qFSR4 was mapped to a 38-kb region containing three open reading frames including the previously characterized NARROW LEAF 1 (NAL1) gene. NAL1, which controls leaf width and also affects vein patterning and polar auxin transport, is the most promising candidate genes for qFSR4. Our results underscore the importance of the development of NILs to confirm the identification of QTL affecting complex traits such as drought resistance.

Genome-wide identification of BURP domain-containing genes in rice reveals a gene family with diverse structures and responses to abiotic stresses.

Increasing evidence suggests that a gene family encoding proteins containing BURP domains have diverse functions in plants, but systematic characterization of this gene family have not been reported. In this study, 17 BURP family genes (OsBURP01-17) were identified and analyzed in rice (Oryza sativa L.). These genes have diverse exon-intron structures and distinct organization of putative motifs. Based on the phylogenetic analysis of BURP protein sequences from rice and other plant species, the BURP family was classified into seven subfamilies, including two subfamilies (BURP V and BURP VI) with members from rice only and one subfamily (BURP VII) with members from monocotyledons only. Two BURP gene clusters, belonging to BURP V and BURP VI, were located in the duplicated region on chromosome 5 and 6 of rice, respectively. Transcript level analysis of BURP genes of rice in various tissues and organs revealed different tempo-spatial expression patterns, suggesting that these genes may function at different stages of plant growth and development. Interestingly, all the genes of the BURP VII subfamily were predominantly expressed in flower organs. We also investigated the expression patterns of BURP genes of rice under different stress conditions. The results suggested that, except for two genes (OsBURP01 and OsBURP13), all other members were induced by at least one of the stresses including drought, salt, cold, and abscisic acid treatment. Two genes (OsBURP05 and OsBURP16) were responsive to all the stress treatments and most of the OsBURP genes were responsive to salt stress. Promoter sequence analysis revealed an over-abundance of stress-related cis-elements in the stress-responsive genes. The data presented here provide important clues for elucidating the functions of genes of this family.