Overexpression of OsACL5 triggers environmentally-dependent leaf rolling and reduces grain size in rice.

Major polyamines include putrescine, spermidine, spermine and thermospermine, which play vital roles in growth and adaptation against environmental changes in plants. Thermospermine (T-Spm) is synthetised by ACL5. The function of ACL5 in rice is still unknown. In this study, we used a reverse genetic strategy to investigate the biological function of OsACL5. We generated several knockout mutants by pYLCRISPR/Cas9 system and overexpressing (OE) lines of OsACL5. Interestingly, the OE plants exhibited environmentally-dependent leaf rolling, smaller grains, lighter 1000-grain weight and reduction in yield per plot. The area of metaxylem vessels of roots and leaves of OE plants were significantly smaller than those of WT, which possibly caused reduction in leaf water potential, resulting in leaf rolling with rise in the environmental temperature and light intensity and decrease in humidity. Additionally, the T-Spm contents were markedly increased by over ninefold whereas the ethylene evolution was reduced in OE plants, suggesting that T-Spm signalling pathway interacts with ethylene pathway to regulate multiple agronomic characters. Moreover, the osacl5 exhibited an increase in grain length, 1000-grain weight, and yield per plot. OsACL5 may affect grain size via mediating the expression of OsDEP1, OsGS3 and OsGW2. Furthermore, haplotypes analysis indicated that OsACL5 plays a conserved function on regulating T-Spm levels during the domestication of rice. Our data demonstrated that identification of OsACL5 provides a theoretical basis for understanding the physiological mechanism of T-Spm which may play roles in triggering environmentally dependent leaf rolling; OsACL5 will be an important gene resource for molecular breeding for higher yield.

Dietary potential of the symbiotic fungus Penicillium herquei for the larvae of a nonsocial fungus-cultivating weevil Euops chinensis.

Many insect taxa cultivate fungi for food. Compared to well-known fungus cultivation in social insects, our knowledge on fungus cultivation in nonsocial insects is still limited. Here, we studied the nutritional potentials of the fungal cultivar, Penicillium herquei, for the larvae of its nonsocial insect farmer, Euops chinensis, a specialist on Japanese knotweed Reynoutria japonica. Overall, fungal hyphae and leaf rolls contained significantly higher carbon (C), stable isotopes of C (δ13C), and nitrogen (δ15N) but significantly lower C/N ratios compared to unrolled leaves, whereas insect bodies contained significantly higher N contents but lower C and C/N ratios compared to other types of samples. The MixSIAR model indicated that fungal hyphae contributed a larger proportion (0.626-0.797) to the diet of E. chinensis larvae than leaf materials. The levels of ergosterol, six essential amino acids, seven nonessential amino acids, and three B vitamins tested in fungal hyphae and/or leaf rolls were significantly higher than in unrolled leaves and/or larvae. The P. herquei genome contains the complete set of genes required for the biosynthesis of ergosterol, the essential amino acids valine and threonine, nine nonessential amino acids, and vitamins B2 and B3, whereas some genes associated with five essential and one nonessential amino acid were lost in the P. herquei genome. These suggest that P. herquei is capable of providing the E. chinensis larvae food with ergosterol, amino acids, and B vitamins. P. herquei appears to be able to synthesize or concentrate these nutrients considering that they were specifically concentrated in fungal hyphae. IMPORTANCE: The cultivation of fungi for food has occurred across divergent insect lineages such as social ants, termites, and ambrosia beetles, as well as some seldom-reported solitary insects. Although the fungal cultivars of these insects have been studied for decades, the dietary potential of fungal cultivars for their hosts (especially for those nonsocial insects) is largely unknown. Our research on the mutualistic system Euops chinensis-Penicillium herquei represents an example of the diverse nutritional potentials of the fungal cultivar P. herquei in the diet of the larvae of its solitary host, E. chinensis. These results demonstrate that P. herquei has the potential to synthesize or concentrate ergosterol, amino acids, and B vitamins and benefits the larvae of E. chinensis. Our findings would shed light on poorly understood fungal cultivation mutualisms in nonsocial insects and underscore the nutritional importance of fungal cultivars in fungal cultivation mutualisms.

Leaf wettability and leaf angle affect air-moisture deposition in wheat for self-irrigation.

BACKGROUND: Climate change and depleting water sources demand scarce natural water supplies like air moisture to be used as an irrigation water source. Wheat production is threatened by the climate variability and extremes climate events especially heat waves and drought. The present study focused to develop the wheat plant for self-irrigation through optimizing leaf architecture and surface properties for precise irrigation. METHODS: Thirty-four genotypes were selected from 1796 genotypes with all combinations of leaf angle and leaf rolling. These genotypes were characterized for morpho-physiological traits and soil moisture content at stem-elongation and booting stages. Further, a core set of ten genotypes was evaluated for stem flow efficiency and leaf wettability. RESULTS: Biplot, heat map, and correlation analysis indicated wide diversity and traits association. The environmental parameters indicated substantial amount of air moisture (> 60% relative humidity) at the critical wheat growth stages. Leaf angle showed negative association with leaf rolling, physiological and yield traits, adaxial and abaxial contact angle while leaf angle showed positive association with the stem flow water. The wettability and air moisture harvesting indicated that the genotypes (coded as 1, 7, and 18) having semi-erect to erect leaf angle, spiral rolling, and hydrophilic leaf surface (<90o) with contact angle hysteresis less than 10o had higher soil moisture content (6-8%) and moisture harvesting efficiency (3.5 ml). CONCLUSIONS: These findings can provide the basis to develop self-irrigating, drought-tolerant wheat cultivars as an adaptation to climate change.

Leaf rolling precedes stomatal closure in rice (Oryza sativa) under drought conditions.

Leaf rolling is a physiological response to drought that may help to reduce water loss, but its significance as a contribution to drought tolerance is uncertain. We scored the leaf rolling of four rice genotypes along an experimental drought gradient using an improved cryo-microscopy method. Leaf water potential (Ψleaf), gas exchange, chlorophyll fluorescence, leaf hydraulic conductance, rehydration capacity, and the bulk turgor loss point were also analysed. During the drought process, stomatal conductance declined sharply to reduce water loss, and leaves rolled up before the stomata completely closed. The leaf water loss rate of rolled leaves was significantly reduced compared with artificially flattened leaves. The Ψleaf threshold of initial leaf rolling ranged from -1.95 to -1.04 MPa across genotypes. When a leaf rolled so that the leaf edges were touching, photosynthetic rate and stomatal conductance declined more than 80%. Across genotypes, leaf hydraulic conductance declined first, followed by gas exchange and chlorophyll fluorescence parameters. However, the Ψleaf threshold for a given functional trait decline differed significantly among genotypes, with the exception of leaf hydraulic conductance. Our results suggested that leaf rolling was mechanistically linked to drought avoidance and tolerance traits and might serve as a useful phenotypic trait for rice breeding in future drought scenarios.

Maize WRKY28 interacts with the DELLA protein D8 to affect skotomorphogenesis and participates in the regulation of shade avoidance and plant architecture.

Competition for light from neighboring vegetation can trigger the shade-avoidance response (SAR) in plants, which is detrimental to their yield. The molecular mechanisms regulating SAR are well established in Arabidopsis, and some regulators of skotomorphogenesis have been found to be involved in the regulation of the SAR and plant architecture. However, the role of WRKY transcription factors in this process has rarely been reported, especially in maize (Zea mays). Here, we report that maize Zmwrky28 mutants exhibit shorter mesocotyls in etiolated seedlings. Molecular and biochemical analyses demonstrate that ZmWRKY28 directly binds to the promoter regions of the Small Auxin Up RNA (SAUR) gene ZmSAUR54 and the Phytochrome-Interacting Factor (PIF) gene ZmPIF4.1 to activate their expression. In addition, the maize DELLA protein Dwarf Plant8 (D8) interacts with ZmWRKY28 in the nucleus to inhibit its transcriptional activation activity. We also show that ZmWRKY28 participates in the regulation of the SAR, plant height, and leaf rolling and erectness in maize. Taken together, our results reveal that ZmWRKY28 is involved in GA-mediated skotomorphogenic development and can be used as a potential target to regulate SAR for breeding of high-density-tolerant cultivars.

Improving Rice Leaf Shape Using CRISPR/Cas9-Mediated Genome Editing of SRL1 and Characterizing Its Regulatory Network Involved in Leaf Rolling through Transcriptome Analysis.

Leaf rolling is a crucial agronomic trait to consider in rice (Oryza sativa L.) breeding as it keeps the leaves upright, reducing interleaf shading and improving photosynthetic efficiency. The SEMI-ROLLED LEAF 1 (SRL1) gene plays a key role in regulating leaf rolling, as it encodes a glycosylphosphatidylinositol-anchored protein located on the plasma membrane. In this study, we used CRISPR/Cas9 to target the second and third exons of the SRL1 gene in the indica rice line GXU103, which resulted in the generation of 14 T0 transgenic plants with a double-target mutation rate of 21.4%. After screening 120 T1 generation plants, we identified 26 T-DNA-free homozygous double-target mutation plants. We designated the resulting SRL1 homozygous double-target knockout as srl1-103. This line exhibited defects in leaf development, leaf rolling in the mature upright leaves, and a compact nature of the fully grown plants. Compared with the wild type (WT), the T2 generation of srl1-103 varied in two key aspects: the width of flag leaf (12.6% reduction compared with WT) and the leaf rolling index (48.77% increase compared with WT). In order to gain a deeper understanding of the involvement of SRL1 in the regulatory network associated with rice leaf development, we performed a transcriptome analysis for the T2 generation of srl1-103. A comparison of srl1-103 with WT revealed 459 differentially expressed genes (DEGs), including 388 upregulated genes and 71 downregulated genes. In terms of the function of the DEGs, there seemed to be a significant enrichment of genes associated with cell wall synthesis (LOC_Os08g01670, LOC_Os05g46510, LOC_Os04g51450, LOC_Os10g28080, LOC_Os04g39814, LOC_Os01g71474, LOC_Os01g71350, and LOC_Os11g47600) and vacuole-related genes (LOC_Os09g23300), which may partially explain the increased leaf rolling in srl1-103. Furthermore, the significant downregulation of BAHD acyltransferase-like protein gene (LOC_Os08g44840) could be the main reason for the decreased leaf angle and the compact nature of the mutant plants. In summary, this study successfully elucidated the gene regulatory network in which SRL1 participates, providing theoretical support for targeting this gene in rice breeding programs to promote variety improvement.

ESCRT-III component OsSNF7.2 modulates leaf rolling by trafficking and endosomal degradation of auxin biosynthetic enzyme OsYUC8 in rice.

The endosomal sorting complex required for transport (ESCRT) is highly conserved in eukaryotic cells and plays an essential role in the biogenesis of multivesicular bodies and cargo degradation to the plant vacuole or lysosomes. Although ESCRT components affect a variety of plant growth and development processes, their impact on leaf development is rarely reported. Here, we found that OsSNF7.2, an ESCRT-III component, controls leaf rolling in rice (Oryza sativa). The Ossnf7.2 mutant rolled leaf 17 (rl17) has adaxially rolled leaves due to the decreased number and size of the bulliform cells. OsSNF7.2 is expressed ubiquitously in all tissues, and its protein is localized in the endosomal compartments. OsSNF7.2 homologs, including OsSNF7, OsSNF7.3, and OsSNF7.4, can physically interact with OsSNF7.2, but their single mutation did not result in leaf rolling. Other ESCRT complex subunits, namely OsVPS20, OsVPS24, and OsBRO1, also interact with OsSNF7.2. Further assays revealed that OsSNF7.2 interacts with OsYUC8 and aids its vacuolar degradation. Both Osyuc8 and rl17 Osyuc8 showed rolled leaves, indicating that OsYUC8 and OsSNF7.2 function in the same pathway, conferring leaf development. This study reveals a new biological function for the ESCRT-III components, and provides new insights into the molecular mechanisms underlying leaf rolling.

Non-rolling flag leaves use an effective mechanism to reduce water loss and light-induced damage under drought stress.

BACKGROUND AND AIMS: The study reports on four different types of flag leaf rolling under soil drought in relation to the level of cell wall-bound phenolics. The flag leaf colonization by aphids, as a possible bioindicator of the accumulation of cell wall-bound phenolics, was also estimated. METHODS: The proteins of the photosynthetic apparatus that form its core and are crucial for maintaining its stability (D1/PsbA protein), limit destructive effects of light (PsbS, a protein binding carotenoids in the antennas) and participate in efficient electron transport between photosystems II (PSII) and PSI (Rieske iron-sulfur protein of the cytochrome b6f complex) were evaluated in two types of flag leaf rolling. Additionally, biochemical and physiological reactions to drought stress in rolling and non-rolling flag leaves were compared. KEY RESULTS: The study identified four types of genome-related types of flag leaf rolling. The biochemical basis for these differences was a different number of phenolic molecules incorporated into polycarbohydrate structures of the cell wall. In an extreme case of non-rolling dehydrated flag leaves, they were found to accumulate high amounts of cell wall-bound phenolics that limited cell water loss and protected the photosynthetic apparatus against excessive light. PSII was also additionally protected against excess light by the accumulation of photosynthetic apparatus proteins that ensured stable and efficient transport of excitation energy beyond PSII and its dissipation as far-red fluorescence and heat. Our analysis revealed a new type of flag leaf rolling brought about by an interaction between wheat and rye genomes, and resulting in biochemical specialization of flexible, rolling and rigid, non-rolling parts of the flag leaf. The study confirmed limited aphid colonization of the flag leaves with enhanced content of cell wall-bound phenolics. CONCLUSIONS: Non-rolling leaves developed effective adaptation mechanisms to reduce both water loss and photoinhibitory damage to the photosynthetic apparatus under drought stress.

DRB2 Modulates Leaf Rolling by Regulating Accumulation of MicroRNAs Related to Leaf Development in Rice.

As an important agronomic trait in rice (Oryza sativa), moderate leaf rolling helps to maintain the erectness of leaves and minimize shadowing between leaves, leading to improved photosynthetic efficiency and grain yield. However, the molecular mechanisms underlying rice leaf rolling still need to be elucidated. Here, we isolated a rice mutant, rl89, showing adaxially rolled leaf phenotype due to decreased number and size of bulliform cells. We confirmed that the rl89 phenotypes were caused by a single nucleotide substitution in OsDRB2 (LOC_Os10g33970) gene encoding DOUBLE-STRANDED RNA-BINDING2. This gene was constitutively expressed, and its encoded protein was localized to both nucleus and cytoplasm. Yeast two-hybrid assay showed that OsDRB2 could interact with DICER-LIKE1 (DCL1) and OsDRB1-2 respectively. qRT-PCR analysis of 29 related genes suggested that defects of the OsDRB2-miR166-OsHBs pathway could play an important role in formation of the rolled leaf phenotype of rl89, in which OsDRB2 mutation reduced miR166 accumulation, resulting in elevated expressions of the class III homeodomain-leucine zipper genes (such as OsHB1, 3 and 5) involved in leaf polarity and/or morphology development. Moreover, OsDRB2 mutation also reduced accumulation of miR160, miR319, miR390, and miR396, which could cause the abnormal leaf development in rl89 by regulating expressions of their target genes related to leaf development.

Heterodimer formed by ROC8 and ROC5 modulates leaf rolling in rice.

Moderately rolled leaf is one of the target traits of the ideal plant architecture in rice breeding. Many genes, including homeodomain leucine zipper IV transcription factors ROC5 and ROC8, regulating rice leaf rolling have been cloned and functionally analysed. However, the molecular mechanism by which these genes modulate leaf-rolling remains largely elusive. In this study, we demonstrated the transcription activation activity of both ROC8 and ROC5. Overexpressing ROC8 caused adaxially rolled leaves due to decreased number and size of bulliform cells, whereas knockout of ROC8 induced abaxially rolled leaves due to increased number and size of bulliform cells. ROC8 and ROC5 each could form homodimer, but ROC8 interacted preferably with ROC5 to forms a heterodimer. Importantly, we showed that the ROC8-ROC5 heterodimer rather than the homodimer of ROC8 or ROC5 was functional as neither overexpressing ROC8 in the ROC5 mutant nor overexpressing ROC5 in the ROC8-knockout line could rescue the mutant phenotype. This was further partially supported by the identification of a large number of common differentially expressed genes in single and double mutants of roc8 and roc5. ROC8 and ROC5 were functionally additive as the phenotype of abaxially rolled leaves was stronger in the roc5roc8 double mutant than in their single mutants. Our results provide evidence for the role of dimerization of ROC members in regulating leaf rolling of rice.

Combining UAV-RGB high-throughput field phenotyping and genome-wide association study to reveal genetic variation of rice germplasms in dynamic response to drought stress.

Accurate and high-throughput phenotyping of the dynamic response of a large rice population to drought stress in the field is a bottleneck for genetic dissection and breeding of drought resistance. Here, high-efficiency and high-frequent image acquisition by an unmanned aerial vehicle (UAV) was utilized to quantify the dynamic drought response of a rice population under field conditions. Deep convolutional neural networks (DCNNs) and canopy height models were applied to extract highly correlated phenotypic traits including UAV-based leaf-rolling score (LRS_uav), plant water content (PWC_uav) and a new composite trait, drought resistance index by UAV (DRI_uav). The DCNNs achieved high accuracy (correlation coefficient R = 0.84 for modeling set and R = 0.86 for test set) to replace manual leaf-rolling rating. PWC_uav values were precisely estimated (correlation coefficient R = 0.88) and DRI_uav was modeled to monitor the drought resistance of rice accessions dynamically and comprehensively. A total of 111 significantly associated loci were detected by genome-wide association study for the three dynamic traits, and 30.6% of them were not detected in previous mapping studies using nondynamic drought response traits. Unmanned aerial vehicle and deep learning are confirmed effective phenotyping techniques for more complete genetic dissection of rice dynamic responses to drought and exploration of valuable alleles for drought resistance improvement.

Leaf morphology, rather than plant water status, underlies genetic variation of rice leaf rolling under drought.

Soil drying causes leaf rolling in rice, but the relationship between leaf rolling and drought tolerance has historically confounded selection of drought-tolerant genotypes. In this study on tropical japonica and aus diversity panels (170-220 genotypes), the degree of leaf rolling under drought was more affected by leaf morphology than by stomatal conductance, leaf water status, or maintenance of shoot biomass and grain yield. A range of canopy temperature and leaf rolling (measured as change in normalized difference vegetation index [ΔNDVI]) combinations were observed among aus genotypes, indicating that some genotypes continued transpiration while rolled. Association mapping indicated colocation of genomic regions for leaf rolling score and ΔNDVI under drought with previously reported leaf rolling genes and gene networks related to leaf anatomy. The relatively subtle variation across these large diversity panels may explain the lack of agreement of this study with earlier reports that used small numbers of genotypes that were highly divergent in hydraulic traits driving leaf rolling differences. This study highlights the large range of physiological responses to drought among rice genotypes and emphasizes that drought response processes should be understood in detail before incorporating them into a varietal selection programme.

Histological, Physiological, and Comparative Proteomic Analyses Provide Insights into Leaf Rolling in Brassica napus.

Moderate leaf rolling is important in ideotype breeding, as it improves photosynthetic efficiency and therefore increases crop yields. To understand the regulatory network of leaf rolling in Brassica napus, a down-curved leaf mutant ( Bndcl1) has been investigated. Physiological analyses indicated that the chlorophyll contents and antioxidant enzyme activities were remarkably increased and the photosynthetic performance was significantly improved in Bndcl1. Consistent with these findings, 943 differentially accumulated proteins (DAPs) were identified in the Bndcl1 mutant and its wild-type plants using iTRAQ-based comparative proteomic analyses. Enrichment analysis of proteins with higher abundance in Bndcl1 revealed that the functional category "photosynthesis" was significantly overrepresented. Moreover, proteins associated with oxidative stress response and photosystem II repairing were also up-accumulated in Bndcl1, which might help the mutant to sustain the photosynthetic efficiency under unfavorable conditions. Histological observation showed that the mutant displayed defects in adaxial-abaxial patterning. Important DAPs associated with leaf polarity establishment were detected in Bndcl1, including ribosomal proteins, proteins involved in post-transcriptional gene silencing, and proteins related to brassinosteroid. Together, our findings may help clarify the mechanisms underlying leaf rolling and its physiological effects on plants and may facilitate ideotype breeding in Brassica napus.

CLD1/SRL1 modulates leaf rolling by affecting cell wall formation, epidermis integrity and water homeostasis in rice.

Leaf rolling is considered as one of the most important agronomic traits in rice breeding. It has been previously reported that SEMI-ROLLED LEAF 1 (SRL1) modulates leaf rolling by regulating the formation of bulliform cells in rice (Oryza sativa); however, the regulatory mechanism underlying SRL1 has yet to be further elucidated. Here, we report the functional characterization of a novel leaf-rolling mutant, curled leaf and dwarf 1 (cld1), with multiple morphological defects. Map-based cloning revealed that CLD1 is allelic with SRL1, and loses function in cld1 through DNA methylation. CLD1/SRL1 encodes a glycophosphatidylinositol (GPI)-anchored membrane protein that modulates leaf rolling and other aspects of rice growth and development. The cld1 mutant exhibits significant decreases in cellulose and lignin contents in secondary cell walls of leaves, indicating that the loss of function of CLD1/SRL1 affects cell wall formation. Furthermore, the loss of CLD1/SRL1 function leads to defective leaf epidermis such as bulliform-like epidermal cells. The defects in leaf epidermis decrease the water-retaining capacity and lead to water deficits in cld1 leaves, which contribute to the main cause of leaf rolling. As a result of the more rapid water loss and lower water content in leaves, cld1 exhibits reduced drought tolerance. Accordingly, the loss of CLD1/SRL1 function causes abnormal expression of genes and proteins associated with cell wall formation, cuticle development and water stress. Taken together, these findings suggest that the functional roles of CLD1/SRL1 in leaf-rolling regulation are closely related to the maintenance of cell wall formation, epidermal integrity and water homeostasis.

The wheat MYB transcription factor TaMYB18 regulates leaf rolling in rice.

Leaf rolling is an important agronomic trait in crop breeding. Moderate leaf rolling maintains the erectness of leaves and minimizes shadowing between leaves, leading to improved photosynthetic efficiency. Although some genes controlling leaf rolling have been isolated from rice and other plant species, few studies have examined leaf rolling in wheat. In the present study, the leaf rolling regulator gene, TaMYB18, was identified in a large-scale transgene project involving the transformation of 1455 wheat transcription factor genes into rice. Three homologous sequences of TaMYB18 were isolated from hexaploid wheat and localized to chromosomes 5A, 5B and 5D, respectively. TaMYB18, an R2R3-MYB transcription factor, localized to the nucleus. TaMYB18 overexpression induced leaf rolling in transgenic rice. Additionally, the three members of TaMYB18 exhibited functional redundancy in rice. Furthermore, the function of TaMYB18 in regulating leaf rolling in rice was a dose-dependent. Taken together, these results indicate that TaMYB18 may play an important role in the regulation of leaf development.

Semi-Rolled Leaf2 modulates rice leaf rolling by regulating abaxial side cell differentiation.

Moderate leaf rolling maintains the erectness of leaves and minimizes the shadowing between leaves which is helpful to establish ideal plant architecture. Here, we describe asrl2(semi-rolled leaf2) rice mutant, which has incurved leaves due to the presence of defective sclerenchymatous cells on the abaxial side of the leaf and displays narrow leaves and reduced plant height. Map-based cloning revealed that SRL2 encodes a novel plant-specific protein of unknown biochemical function.SRL2 was mainly expressed in the vascular bundles of leaf blades, leaf sheaths, and roots, especially in their sclerenchymatous cells. The transcriptional activities of several leaf development-related YABBY genes were significantly altered in the srl2 mutant. Double mutant analysis suggested that SRL2 and SHALLOT-LIKE1(SLL1)/ROLLED LEAF9(RL9) function in distinct pathways that regulate abaxial-side leaf development. Hence, SRL2 plays an important role in regulating leaf development, particularly during sclerenchymatous cell differentiation.

The SlARF4-SlHB8 regulatory module mediates leaf rolling in tomato.

Leaf is the main photosynthetic organ in plants and the primary energy source all along the plant life. Given the beneficial role of leaf rolling in improving photosynthetic efficiency and yield in specific environmental conditions, a better understanding of the factors and molecular mechanisms underlying this process is highly suited. Previously, the SlARF4 knocking out mutant exhibited upward curly leaf showed higher resistance to water deficit which driving us to uncover the function of SlARF4 in regulating the curly leaf formation. In this study, we unraveled the unexplored role of the SlARF4-SlHB8 module of transcription factors in the development of leaf rolling. Both SlARF4 loss-of-function and SlHB8 overexpressing tomato plants exhibited upward-rolled leaves, reflecting the active role of the two genes in controlling leaf rolling. Dual-luciferase reporter assays and phenotypic analysis of hybrid progenies suggested that SlHB8 acts downstream of SlARF4 in curly leaf formation. SlARF4 and SlHB8 influence the development of leaf palisade tissues via modulating the expression of genes associated with curly leaf formation. SEM analysis revealed no significant differences in leaf epidermal cells between the two leaf-rolling mutants and the wild type, indicating that curly leaves of arf4 and SlHB8-OE do not result from the asymmetric leaf epidermal cell growth. Our data provide novel insight into the molecular mechanism of abaxial-adaxial determination involving SlARF4 and SlHB8 and reveals that leaf rolling operates via different regulation mechanisms in tomato and Arabidopsis model plant.

Xylella fastidiosa subsp. pauca, Neofusicoccum spp. and the Decline of Olive Trees in Salento (Apulia, Italy): Comparison of Symptoms, Possible Interactions, Certainties and Doubts.

Xylella fastidiosa subsp. pauca (XFP), Neofusicoccum mediterraneum, N. stellenboschiana and other fungi have been found in olive groves of Salento (Apulia, Italy) that show symptoms of severe decline. XFP is well known to be the cause of olive quick decline syndrome (OQDS). It has also been assessed that Neofusicoccum spp. causes a distinct disease syndrome, namely, branch and twig dieback (BTD). All these phytopathogens incite severe symptoms that can compromise the viability of large canopy sectors or the whole tree. However, their specific symptoms are not easily distinguished, especially during the final stages of the disease when branches are definitively desiccated. By contrast, they can be differentiated during the initial phases of the infection when some facets of the diseases are typical, especially wood discoloration, incited solely by fungi. Here, we describe the typical symptomatological features of OQDS and BTD that can be observed in the field and that have been confirmed by Koch postulate experiments. Similar symptoms, caused by some abiotic adverse conditions and even by additional biotic factors, are also described. Thus, this review aims at: (i) raising the awareness that declining olive trees in Salento do not have to be linked a priori to XFP; (ii) defining the guidelines for a correct symptomatic diagnosis to orient proper laboratory analyses, which is crucial for the application of effective control measures. The possibility that bacterium and fungi could act as a polyspecies and in conjunction with predisposing abiotic stresses is also widely discussed.

Increased dehydrin level decreases leaf rolling grade by altering the reactive oxygen species homeostasis and abscisic acid content in maize subjected to osmotic stress.

Dehydrins (DHNs) are stress proteins involved in the development of protective reactions in plants against dehydration. The relationship between DHNs and morphological responses such as leaf rolling in plants exposed to water deficit is not well known. In this study, we detected how variations in DHN levels affect the leaf rolling response in maize exposed to osmotic stress in relation to the antioxidant system and ABA level. In this context, we altered the DHN levels in maize seedlings by treatment with bio-regulators (salicylic acid and abscisic acid) under PEG6000-free and PEG6000-induced osmotic stress. When the DHN levels were increased by the bio-regulators (25 µM SA and 100 µM ABA), the relative expression level of the Zea mays dehydrin COR410 gene increased in the seedlings, while reactive oxygen species (ROS) and leaf rolling grade decreased. Moreover, induction of DHNs caused increases in the antioxidant enzyme activity and content of antioxidant substances, and very high amounts of endogenous abscisic acid. When DHN level was suppressed by a bio-regulator (200 µM SA) in the maize seedlings, dehydrin COR410 expression level decreased, while ROS and the leaf rolling grade increased. Moreover, the antioxidant enzyme activity and content of antioxidant substances decreased in the seedlings, while the amount of abscisic acid increased. Taken all together, an increase in DHN level by bio-regulator treatment can stimulate the antioxidant system, enable abscisic acid regulation, and thus reduce leaf rolling through decreased ROS levels. The results also indicated that DHNs may be involved in the signal pathways inducing expression of some genes related to leaf rolling response, possibly by modulating ROS levels, in maize seedlings exposed to osmotic stress.

Leaf rolling in bread wheat (Triticum aestivum L.) is controlled by the upregulation of a pair of closely linked/duplicate zinc finger homeodomain class transcription factors during moisture stress conditions.

Zinc finger-homeodomain (ZF-HDs) class IV transcriptional factors (TFs) is a plant-specific transcription factor and play a key role in stress responses, plant growth, development, and hormonal signaling. In this study, two new leaf rolling TFs genes, namely TaZHD1 and TaZHD10, were identified in wheat using comparative genomic analysis of the target region that carried a major QTL for leaf rolling identified through multi-environment phenotyping and high throughput genotyping of a RIL population. Structural and functional annotation of the candidate ZHD genes with its closest rice orthologs reflects the species-specific evolution and, undoubtedly, validates the notions of remote-distance homology concept. Meanwhile, the morphological analysis resulted in contrasting difference for leaf rolling in extreme RILs between parental lines HD2012 and NI5439 at booting and heading stages. Transcriptome-wide expression profiling revealed that TaZHD10 transcripts showed significantly higher expression levels than TaZHD1 in all leaf tissues upon drought stress. The relative expression of these genes was further validated by qRT-PCR analysis, which also showed consistent results across the studied genotypes at the booting and anthesis stage. The contrasting modulation of these genes under drought conditions and the available evidenced for its epigenetic behavior that might involve the regulation of metabolic and gene regulatory networks. Prediction of miRNAs resulted in five Tae-miRs that could be associated with RNAi mediated control of TaZHD1 and TaZHD10 putatively involved in the metabolic pathway controlling rolled leaf phenotype. Gene interaction network analysis indicated that TaZHD1 and TaZHD10 showed pleiotropic effects and might also involve other functions in wheat in addition to leaf rolling. Overall, the results increase our understanding of TaZHD genes and provide valuable information as robust candidate genes for future functional genomics research aiming for the breeding of wheat varieties tolerant to leaf rolling.