TaKLU Plays as a Time Regulator of Leaf Growth via Auxin Signaling.

The growth of leaves is subject to strict time regulation. Several genes influencing leaf growth have been identified, but little is known about how genes regulate the orderly initiation and growth of leaves. Here, we demonstrate that TaKLU/TaCYP78A5 contributes to a time regulation mechanism in leaves from initiation to expansion. TaKLU encodes the cytochrome P450 CYP78A5, and its homolog AtKLU has been described whose deletion is detrimental to organ growth. Our results show that TaKLU overexpression increases leaf size and biomass by altering the time of leaf initiation and expansion. TaKLU-overexpressing plants have larger leaves with more cells. Further dynamic observations indicate that enlarged wheat leaves have experienced a longer expansion time. Different from AtKLU inactivation increases leaf number and initiation rates, TaKLU overexpression only smooths the fluctuations of leaf initiation rates by adjusting the initiation time of local leaves, without affecting the overall leaf number and initiation rates. In addition, complementary analyses suggest TaKLU is functionally conserved with AtKLU in controlling the leaf initiation and size and may involve auxin accumulation. Our results provide a new insight into the time regulation mechanisms of leaf growth in wheat.

Strigolactones (SLs) modulate the plastochron by regulating KLUH (KLU) transcript abundance in Arabidopsis.

The timing of leaf emergence at the shoot apical meristem, or plastochron, is highly regulated in plants. Among the genes known to regulate the plastochron in Arabidopsis (Arabidopsis thaliana), KLUH (KLU), orthologous to the rice (Oryza sativa) PLASTOCHRON1, encodes the cytochrome P450 CYP78A5, and is thought to act through generation of a still unknown mobile signal. As klu mutants display not only a short plastochron but also a branching phenotype reminiscent of strigolactone (SL) mutants, we investigated whether KLU/CYP78A5 is involved in SL biosynthesis. We combined a genetic approach, a parasitic plant seed germination bioassay to test klu root exudates, and analysis of transcript abundances of SL-biosynthesis genes in the Arabidopsis klu mutants. We demonstrate that KLU is not involved in the SL-biosynthesis pathway. Moreover, this work allowed us to uncover a new role for SL during Arabidopsis development in modulating plastochron via a KLU-dependent pathway. Globally our data reveal that KLU is required for plastochron-specific SL responses, a first indication of crosstalk between SL and the KLU-derived signal.

A CACTA-like transposable element in the upstream region of BnaA9.CYP78A9 acts as an enhancer to increase silique length and seed weight in rapeseed.

Rapeseed (Brassica napus L.) is a model plant for polyploid crop research and the second-leading source of vegetable oil worldwide. Silique length (SL) and seed weight are two important yield-influencing traits in rapeseed. Using map-based cloning, we isolated qSLWA9, which encodes a P450 monooxygenase (BnaA9.CYP78A9) and functions as a positive regulator of SL. The expression level of BnaA9.CYP78A9 in silique valves of the long-silique variety is much higher than that in the regular-silique variety, which results in elongated cells and a prolonged phase of silique elongation. Plants of the long-silique variety and transgenic plants with high expression of BnaA9.CYP78A9 had a higher concentration of auxin in the developing silique; this induced a number of auxin-related genes but no genes in well-known auxin biosynthesis pathways, suggesting that BnaA9.CYP78A9 may influence auxin concentration by affecting auxin metabolism or an unknown auxin biosynthesis pathway. A 3.7-kb CACTA-like transposable element (TE) inserted in the 3.9-kb upstream regulatory sequence of BnaA9.CYP78A9 elevates the expression level, suggesting that the CACTA-like TE acts as an enhancer to stimulate high gene expression and silique elongation. Marker and sequence analysis revealed that the TE in B. napus had recently been introgressed from Brassica rapa by interspecific hybridization. The insertion of the TE is consistently associated with long siliques and large seeds in both B. napus and B. rapa collections. However, the frequency of the CACTA-like TE in rapeseed varieties is still very low, suggesting that this allele has not been widely used in rapeseed breeding programs and would be invaluable for yield improvement in rapeseed breeding.

Establishment of the Embryonic Shoot Meristem Involves Activation of Two Classes of Genes with Opposing Functions for Meristem Activities.

The shoot meristem, a stem-cell-containing tissue initiated during plant embryogenesis, is responsible for continuous shoot organ production in postembryonic development. Although key regulatory factors including KNOX genes are responsible for stem cell maintenance in the shoot meristem, how the onset of such factors is regulated during embryogenesis is elusive. Here, we present evidence that the two KNOX genes STM and KNAT6 together with the two other regulatory genes BLR and LAS are functionally important downstream genes of CUC1 and CUC2, which are a redundant pair of genes that specify the embryonic shoot organ boundary. Combined expression of STM with any of KNAT6, BLR, and LAS can efficiently rescue the defects of shoot meristem formation and/or separation of cotyledons in cuc1cuc2 double mutants. In addition, CUC1 and CUC2 are also required for the activation of KLU, a cytochrome P450-encoding gene known to restrict organ production, and KLU counteracts STM in the promotion of meristem activity, providing a possible balancing mechanism for shoot meristem maintenance. Together, these results establish the roles for CUC1 and CUC2 in coordinating the activation of two classes of genes with opposite effects on shoot meristem activity.

Maternal control of seed weight in rapeseed (Brassica napus L.): the causal link between the size of pod (mother, source) and seed (offspring, sink).

Seed size/weight is one of the key traits related to plant domestication and crop improvement. In rapeseed (Brassica napus L.) germplasm, seed weight shows extensive variation, but its regulatory mechanism is poorly understood. To identify the key mechanism of seed weight regulation, a systematic comparative study was performed. Genetic, morphological and cytological evidence showed that seed weight was controlled by maternal genotype, through the regulation of seed size mainly via cell number. The physiological evidence indicated that differences in the pod length might result in differences in pod wall photosynthetic area, carbohydrates and the final seed weight. We also identified two pleiotropic major quantitative trait loci that acted indirectly on seed weight via their effects on pod length. RNA-seq results showed that genes related to pod development and hormones were significantly differentially expressed in the pod wall; genes related to development, cell division, nutrient reservoir and ribosomal proteins were all up-regulated in the seeds of the large-seed pool. Finally, we proposed a potential seed weight regulatory mechanism that is specific to rapeseed and novel in plants. The results demonstrate a causal link between the size of the pod (mother, source) and the seed (offspring, sink) in rapeseed, which provides novel insight into the maternal control of seed weight and will open a new research field in plants.

Expression of TaCYP78A3, a gene encoding cytochrome P450 CYP78A3 protein in wheat (Triticum aestivum L.), affects seed size.

Several studies have described quantitative trait loci (QTL) for seed size in wheat, but the relevant genes and molecular mechanisms remain largely unknown. Here we report the functional characterization of the wheat TaCYP78A3 gene and its effect on seed size. TaCYP78A3 encoded wheat cytochrome P450 CYP78A3, and was specifically expressed in wheat reproductive organs. TaCYP78A3 activity was positively correlated with the final seed size. Its silencing caused a reduction of cell number in the seed coat, resulting in an 11% decrease in wheat seed size, whereas TaCYP78A3 over-expression induced production of more cells in the seed coat, leading to an 11-48% increase in Arabidopsis seed size. In addition, the cell number in the final seed coat was determined by the TaCYP78A3 expression level, which affected the extent of integument cell proliferation in the developing ovule and seed. Unfortunately, TaCYP78A3 over-expression in Arabidopsis caused a reduced seed set due to an ovule developmental defect. Moreover, TaCYP78A3 over-expression affected embryo development by promoting embryo integument cell proliferation during seed development, which also ultimately affected the final seed size in Arabidopsis. In summary, our results indicated that TaCYP78A3 plays critical roles in influencing seed size by affecting the extent of integument cell proliferation. The present study provides direct evidence that TaCYP78A3 affects seed size in wheat, and contributes to an understanding of the cellular basis of the gene influencing seed development.

Arabidopsis KLU homologue GmCYP78A72 regulates seed size in soybean.

Soybean (Glycine max) is one of the most important crops in the world, and its yield is largely determined by grain weight and grain size. However, the genes that regulate soybean seed size have not been identified. CYP78A, which is highly conserved within terrestrial plants, regulates organ development. In Arabidopsis, AtCYP78A5/KLU has been shown to determine seed size. In the present study, soybean CYP78A72 (GmCYP78A72), one of the orthologs of KLU, was over-expressed in both Arabidopsis and soybean to examine its function in plant development. GmCYP78A72 heterologous expression in Arabidopsis resulted in enlarged sepals, petals, seeds and carpel. Over-expression of GmCYP78A72 in soybean resulted in increased pea size, which is an extremely desirable trait for enhancing productivity. Moreover, knock-down of GmCYP78A72 does not reduce grain size. However, silencing of GmCYP78A57, GmCYP78A70 and GmCYP78A72 genes in triplet reduces the seed size significantly indicating functional redundancy of these three GmCYP78A genes. In conclusion, we investigated the role of CYP78A in soybean seed regulation, and our strategy can be effectively used to engineer large seed traits in soybean varieties as well as other crops.

TaCYP78A5 regulates seed size in wheat (Triticum aestivum).

Seed size is an important agronomic trait and a major component of seed yield in wheat. However, little is known about the genes and mechanisms that determine the final seed size in wheat. Here, we isolated TaCYP78A5, the orthologous gene of Arabidopsis CYP78A5/KLUH in wheat, from wheat cv. Shaan 512 and demonstrated that the expression of TaCYP78A5 affects seed size. TaCYP78A5 encodes the cytochrome P450 (CYP) 78A5 protein in wheat and rescued the phenotype of the Arabidopsis deletion mutant cyp78a5. By affecting the extent of integument cell proliferation in the developing ovule and seed, TaCYP78A5 influenced the growth of the seed coat, which appears to limit seed growth. TaCYP78A5 silencing caused a 10% reduction in cell numbers in the seed coat, resulting in a 10% reduction in seed size in wheat cv. Shaan 512. By contrast, the overexpression of TaCYP78A5 increased the number of cells in the seed coat, resulting in seed enlargement of ~11-35% in Arabidopsis. TaCYP78A5 activity was positively correlated with the final seed size. However, TaCYP78A5 overexpression significantly reduced seed set in Arabidopsis, possibly due to an ovule development defect. TaCYP78A5 also influenced embryo development by promoting embryo integument cell proliferation during seed development. Accordingly, a working model of the influence of TaCYP7A5 on seed size was proposed. This study provides direct evidence that TaCYP78A5 affects seed size and is a potential target for crop improvement.

KLU/CYP78A5, a Cytochrome P450 Monooxygenase Identified via Fox Hunting, Contributes to Cuticle Biosynthesis and Improves Various Abiotic Stress Tolerances.

Acquired osmotolerance after salt stress is widespread among Arabidopsis thaliana (Arabidopsis) accessions. Most salt-tolerant accessions exhibit acquired osmotolerance, whereas Col-0 does not. To identify genes that can confer acquired osmotolerance to Col-0 plants, we performed full-length cDNA overexpression (FOX) hunting using full-length cDNAs of halophyte Eutrema salsugineum, a close relative of Arabidopsis. We identified EsCYP78A5 as a gene that can confer acquired osmotolerance to Col-0 wild-type (WT) plants. EsCYP78A5 encodes a cytochrome P450 monooxygenase and the Arabidopsis ortholog is known as KLU. We also demonstrated that transgenic Col-0 plants overexpressing AtKLU (AtKLUox) exhibited acquired osmotolerance. Interestingly, KLU overexpression improved not only acquired osmotolerance but also osmo-shock, salt-shock, oxidative, and heat-stress tolerances. Under normal conditions, the AtKLUox plants showed growth retardation with shiny green leaves. The AtKLUox plants also accumulated higher anthocyanin levels and developed denser cuticular wax than WT plants. Compared to WT plants, the AtKLUox plants accumulated significantly higher levels of cutin monomers and very-long-chain fatty acids, which play an important role in the development of cuticular wax and membrane lipids. Endoplasmic reticulum (ER) stress induced by osmotic or heat stress was reduced in AtKLUox plants compared to WT plants. These findings suggest that KLU is involved in the cuticle biosynthesis, accumulation of cuticular wax, and reduction of ER stress induced by abiotic stresses, leading to the observed abiotic stress tolerances.

PaCYP78A9, a Cytochrome P450, Regulates Fruit Size in Sweet Cherry (Prunus avium L.).

Sweet cherry (Prunus avium L.) is an important fruit crop in which fruit size is strongly associated with commercial value; few genes associated with fruit size have, however, been identified in sweet cherry. Members of the CYP78A subfamily, a group of important cytochrome P450s, have been found to be involved in controlling seed size and development in Arabidopsis thaliana, rice, soybean, and tomato. However, the influence of CYP78A members in controlling organ size and the underlying molecular mechanisms in sweet cherry and other fruit trees remains unclear. Here, we characterized a P. avium CYP78A gene PaCYP78A9 that is thought to be involved in the regulation of fruit size and organ development using overexpression and silencing approaches. PaCYP78A9 was significantly expressed in the flowers and fruit of sweet cherry. RNAi silencing of PaCYP78A9 produced small cherry fruits and PaCYP78A9 was found to affect fruit size by mediating mesocarp cell proliferation and expansion during fruit growth and development. Overexpression of PaCYP78A9 in Arabidopsis resulted in increased silique and seed size and PaCYP78A9 was found to be highly expressed in the inflorescences and siliques of transgenic plants. Genes related to cell cycling and proliferation were downregulated in fruit from sweet cherry TRV::PaCYP78A9-silencing lines, suggesting that PaCYP78A9 is likely to be an important upstream regulator of cell cycle processes. Together, our findings indicate that PaCYP78A9 plays an essential role in the regulation of cherry fruit size and provide insights into the molecular basis of the mechanisms regulating traits such as fruit size in P. avium.

Toward understanding the role of CYP78A9 during Arabidopsis reproduction.

After fertilization in Arabidopsis, auxin response in ovules triggers fruit development through the stimulation of gibberellin metabolism. In a recent work, we showed that this model could not explain why CYP78A9 overexpression can uncouple these processes. The specific expression pattern of CYP78A9 suggests its involvement during reproductive development. Moreover, controlled pollination showed that CYP78A9 responds to fertilization. The genetic evidence supports the idea that CYP78A9 and its closest paralogs participate in a pathway that control floral organ size and ovule integuments development as denoted by the phenotypes of es1-D overexpression and cyp78a8 cyp78a9 double mutants. Furthermore, according to previous predictions, perturbations in the flavonol biosynthesis pathway were detected in cyp78a9, cyp78a8 cyp78a9 and es1-D mutants. However, they do not cause the observed phenotypes. Our results add new insights into the role of CYP78A9 in plant reproduction and present the first characterization of metabolite differences between mutants in this gene family.

Over-expression of CYP78A98, a cytochrome P450 gene from Jatropha curcas L, increases seed size of transgenic tobacco

Background: Jatropha curcas L. (further referred to as Jatropha), as a rapidly emerging biofuel crop, has attracted worldwide interest. However, Jatropha is still an undomesticated plant, the true potential of this shrub has not yet been fully realized. To explore the potential of Jatropha, breeding and domestication are needed. Seed size is one of the most important traits of seed yield and has been selected since the beginning of agriculture. Increasing the seed size is a main goal of Jatropha domestication for increasing the seed yield, but the genetic regulation of seed size in Jatropha has not been fully understood. Results: We cloned CYP78A98 gene from Jatropha,a homologue of CYP78A5 in Arabidopsis.Wefound that CYP78A98 was highly expressed in male flower, female flower, stem apex, leaf and developing seed. However, its transcripts were hardly detected in root and stem. CYP78A98 protein localized in endoplasmic reticulum (ER) and the hydrophobic domain at the N-terminus was essential for the correct protein localization. Furthermore, INNER NO OUTER promoter (pINO) drove specific overexpression of CYP78A98 in transgenic tobacco seeds resulted in increased seed size and weight, as well as improved seed protein and fatty acid content. Conclusions: The results indicated that CYP78A98 played a role in Jatropha seed size control. This may help us to better understand the genetic regulation of Jatropha seed development, and accelerate the breeding progress of Jatropha.