Characterization of CYP82 genes involved in the biosynthesis of structurally diverse benzylisoquinoline alkaloids in Corydalis yanhusuo.

Benzylisoquinoline alkaloids (BIAs) represent a significant class of secondary metabolites with crucial roles in plant physiology and substantial potential for clinical applications. CYP82 genes are involved in the formation and modification of various BIA skeletons, contributing to the structural diversity of compounds. In this study, Corydalis yanhusuo, a traditional Chinese medicine rich in BIAs, was investigated to identify the catalytic function of CYP82s during BIA formation. Specifically, 20 CyCYP82-encoding genes were cloned, and their functions were identified in vitro. Ten of these CyCYP82s were observed to catalyze hydroxylation, leading to the formation of protopine and benzophenanthridine scaffolds. Furthermore, the correlation between BIA accumulation and the expression of CyCYP82s in different tissues of C. yanhusuo was assessed their. The identification and characterization of CyCYP82s provide novel genetic elements that can advance the synthetic biology of BIA compounds such as protopine and benzophenanthridine, and offer insights into the biosynthesis of BIAs with diverse structures in C. yanhusuo.

Genome-Wide Analysis of Aux/IAA Gene Family in Artemisia argyi: Identification, Phylogenetic Analysis, and Determination of Response to Various Phytohormones.

Artemisia argyi is a traditional herbal medicine plant, and its folium artemisia argyi is widely in demand due to moxibustion applications globally. The Auxin/indole-3-acetic acid (Aux/IAA, or IAA) gene family has critical roles in the primary auxin-response process, with extensive involvement in plant development and stresses, controlling various essential traits of plants. However, the systematic investigation of the Aux/IAA gene family in A. argyi remains limited. In this study, a total of 61 Aux/IAA genes were comprehensively identified and characterized. Gene structural analysis indicated that 46 Aux/IAA proteins contain the four typical domains, and 15 Aux/IAA proteins belong to non-canonical IAA proteins. Collinear prediction and phylogenetic relationship analyses suggested that Aux/IAA proteins were grouped into 13 distinct categories, and most Aux/IAA genes might experience gene loss during the tandem duplication process. Promoter cis-element investigation indicated that Aux/IAA promoters contain a variety of plant hormone response and stress response cis-elements. Protein interaction prediction analysis demonstrated that AaIAA26/29/7/34 proteins are possibly core members of the Aux/IAA family interaction. Expression analysis in roots and leaves via RNA-seq data indicated that the expression of some AaIAAs exhibited tissue-specific expression patterns, and some AaIAAs were involved in the regulation of salt and saline-alkali stresses. In addition, RT-qPCR results indicated that AaIAA genes have differential responses to auxin, with complex response patterns in response to other hormones, indicating that Aux/IAA may play a role in connecting auxin and other hormone signaling pathways. Overall, these findings shed more light on AaIAA genes and offer critical foundational knowledge toward the elucidation of their function during plant growth, stress response, and hormone networking of Aux/IAA family genes in A. argyi.

Multi-omics analysis of small RNA, transcriptome, and degradome to identify putative miRNAs linked to MeJA regulated and oridonin biosynthesis in Isodon rubescens.

Isodon rubescens has garnered much attention due to its anti-tumor or anti-cancer properties. However, little is known about the molecular mechanism of oridonin biosynthesis leveraging the regulatory network between small RNAs and mRNAs. In this study, the regulatory networks of miRNAs and targets were examined by combining mRNA, miRNA, and degradome. A total of 348 miRNAs, including 287 known miRNAs and 61 novel miRNAs, were identified. Among them, 51 miRNAs were significantly expressed, and 36 miRNAs responded to MeJA. A total of 3066 target genes were associated with 228 miRNAs via degradome sequencing. Multi-omics analysis demonstrated that 27 miRNA-mRNA pairs were speculated to be involved in MeJA regulation, and 36 miRNA-mRNA pairs were hypothesized to be involved in the genotype-dependence of I. rubescens. Furthermore, 151 and 7 miRNA-mRNA modules were likely engaged in oridonin biosynthesis as identified by psRNATarget and degradome sequencing, respectively. Some miRNA-mRNA modules were confirmed via RT-qPCR. Moreover, miRNAs targeting plant hormone signal transduction pathway genes were identified, such as miR156, miR167, miR393, and PC-3p-19822_242. Collectively, our results demonstrate for the first time that miRNAs are identified in I. rubescens, and laid a solid foundation for further research on the molecular mechanism of oridonin biosynthesis mediated by miRNA.

PePYL4 enhances drought tolerance by modulating water-use efficiency and ROS scavenging in Populus.

Drought is one of the major limiting factors in the growth of terrestrial plants. Abscisic acid (ABA) and pyrabactin resistance 1/prabactin resistance-1 like/regulatory components of ABA receptors (PYR/PYL/RCARs) play a key role in response to drought stress. However, the underlying mechanisms of this control remain largely elusive in trees. In this study, PePYL4, a potential ortholog of the PYR/PYL/RCARs gene, was cloned from Populus euphratica. It was localized in the cytoplasm and nucleus, induced by ABA, osmotic and dehydration treatments. To study the potential biological functions of PePYL4, transgenic triploid white poplars (Populus tomentosa 'YiXianCiZhu B38') overexpressing PePYL4 were generated. PePYL4 overexpression significantly increased ABA sensitivity and reduced stomatal aperture. Compared with wild-type plants, transgenic plants had higher water-use efficiency (WUE) and lower transpiration. When exposed to drought stress, PePYL4 overexpression plants maintained higher photosynthetic activity and accumulated more biomass. Moreover, overexpression of PePYL4 improved antioxidant enzyme activity and ascorbate content to accelerate reactive oxygen species scavenging. Meanwhile, upregulation expression of the stress-related genes also contributed to improving the drought tolerance of transgenic plants. In conclusion, our data suggest that PePYL4 is a promising gene target for regulating WUE and drought tolerance in Populus.

Validation of suitable reference genes by various algorithms for gene expression analysis in Isodon rubescens under different abiotic stresses.

Isodon rubescens (Hemsley) H. Hara (Lamiaceae) is a traditional Chinese medicine plant that has been used to treat various human diseases. Oridonin is one of the main active ingredients, and the route of its molecular biosynthesis remains to be determined. The study of gene expression patterns can provide clues toward the understanding of its biological functions. The selection of suitable reference genes for normalizing target gene expression is the first steps in any quantitative real-time PCR (RT-qPCR) gene expression study. Therefore, validation of suitable reference genes is necessary for obtaining reliable results in RT-qPCR analyses of I. rubescens. Here, 12 candidate reference genes were chosen, and their expression stability in different tissues of I. rubescens and in leaves under different abiotic stresses (NaCl, dehydration, SA, MeJA, and ABA) was evaluated using the ∆Ct, NormFinder, GeNorm, BestKeeper, and RankAggreg statistical tools. Analysis using the comprehensive tools of RankAggreg algorithm showed that GADPH, 18S and eIF were stably expressed in different tissues; UBQ, Apt, and HIS; Cycl, UBQ, and PP2A; GADPH, 18S, and eIF; eIF, UBQ, and PP2A; TUB, Cycl, and UBQ; were the best three candidate reference genes for the samples of Dehydration, NaCl, SA, MeJA, and ABA treatment, respectively. While for the concatenated sets of ND (NaCl and dehydration) and SMA (SA, MeJA, and ABA), UBQ, HIS, and TUA; UBQ, eIF and Apt were the three appropriate candidate reference genes, respectively. In addition, the expression patterns of HMGR in different tissues and under different treatments were used to confirm the reliability of the selected reference genes, indicating that the use of an inappropriate reference gene as the internal control will cause results with a large deviation. This work is the first study on the expression stability of reference genes in I. rubescens and will be particularly useful for gene functional research in this species.

Molecular Cloning and Functional Analysis of IrUGT86A1-like Gene in Medicinal Plant Isodon rubescens (Hemsl.) Hara.

The synthesis of secondary metabolites in plants often includes glycosylation modifications. Often, the final step of constructing plant secondary metabolites is completed by glycosylation transferases, which are also involved in many cell processes. In this study, a UDP-glycosyltransferase gene (UGT) was amplified from Isodon rubescens (Hemsl.) Hara with RT-PCR and named IrUGT86A1-like (GenBank: MZ913258). Here, we found that IrUGT86A1-like gene is 1450 bp in length and encodes for 479 amino acids. Bioinformatics analysis revealed that IrUGT86A1-like is a stable and hydrophilic protein, located in the cytoplasm with a transmembrane domain. Phylogenetic analysis showed that IrUGT86A1-like protein has the closest genetic relationship with the UDP-glycosyltransferase 86A1-like protein (XP_042054241.1) of Salvia splendens. RT-qPCR analysis demonstrated that the expression of IrUGT86A1-like gene varied in different tissues; leaves had the highest expression followed by flowers, stems, and roots had the lowest expression. This expression trend is similar to the distribution of oridonin content in different tissues of I. rubescens. Additionally, IrUGT86A1-like gene was found to be positively enhanced by NaCl and MeJA treatment, and in contrast was down-regulated by ABA treatment. Finally, the prokaryotic expression vector pEASY®-Blunt E1-IrUGT86A1 was successfully used to express about 53 KD of IrUGT86A1-like protein. This research builds a foundation for further investigation on the function of this gene in the synthesis and modification of secondary metabolites.

Comparative analysis of chloroplast genomes reveals phylogenetic relationships and intraspecific variation in the medicinal plant Isodon rubescens.

Isodon rubescens (Hemsley) H. Hara (Lamiaceae) is a traditional Chinese medicine plant that has been used to treat various human diseases and conditions such as inflammation, respiratory and gastrointestinal bacterial infections, and malignant tumors. However, the contents of the main active components of I. rubescens from different origins differ significantly, which greatly affected its quality. Therefore, a molecular method to identify and classify I. rubescens is needed. Here, we report the DNA sequence of the chloroplast genome of I. rubescens collected from Lushan, Henan province. The genome is 152,642 bp in length and has a conserved structure that includes a pair of IR regions (25,726 bp), a LSC region (83,527 bp) and a SSC region (17,663 bp). The chloroplast genome contains 113 unique genes, four rRNA genes, 30 tRNA genes, and 79 protein-coding genes, 23 of which contain introns. The protein-coding genes account for a total of 24,412 codons, and most of them are A/T biased usage. We identified 32 simple sequence repeats (SSRs) and 48 long repeats. Furthermore, we developed valuable chloroplast molecular resources by comparing chloroplast genomes from three Isodon species, and both mVISTA and DnaSP analyses showed that rps16-trnQ, trnS-trnG, and ndhC-trnM are candidate regions that will allow the identification of intraspecific differences within I. rubescens. Also 14 candidate fragments can be used to identify interspecific differences between species in Isodon. A phylogenetic analysis of the complete chloroplast genomes of 24 species in subfamily Nepetoideae was performed using the maximum likelihood method, and shows that I. rubescens clustered closer to I. serra than I. lophanthoides. Interestingly, our analysis showed that I. rubescens (MW018469.1) from Xianyang, Shaanxi Province (IR-X), is closer to I. serra than to the other two I. rubescens accessions. These results strongly indicate that intraspecific diversity is present in I. rubescens. Therefore, our results provide further insight into the phylogenetic relationships and interspecific diversity of species in the genus Isodon.

The complete chloroplast genome sequence of Rehmannia glutinosa (Gaertn.) DC. Wild. (Rehmannia).

In this study, we constructed and annotated a complete circular chloroplast genome of wild R. glutinosa. The chloroplast genome of wild R. glutinosa is 153,678 bp in length, including two inverted repeat (IR) regions of 25,759 bp, separated by a large single copy (LSC) region of 84,544 bp and a small single copy (SSC) region of 17,616 bp. The genome contains 149 genes, including 104 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Neighbor-joining method phylogenomic analysis showed that wild R. glutinosa formed a monophyletic group, and was sister to other groups of R. glutinosa.

PeSHN1 regulates water-use efficiency and drought tolerance by modulating wax biosynthesis in poplar.

Wax, a hydrophobic structure that provides an effective waterproof barrier to the leaves, is an important drought adaptation trait for preventing water loss. However, limited knowledge exists regarding the molecular mechanisms underlying wax biosynthesis in trees. Here, PeSHN1, an AP2/ethylene response factor transcription factor, was isolated from a fast-growing poplar Populus × euramericana cv. 'Neva' clone. To study the potential biological functions of PeSHN1, transgenic 84K poplar (Populus alba × Populus glandulosa) plants overexpressing PeSHN1 were generated. PeSHN1 overexpression resulted in decreased transpiration, increased water-use efficiency (WUE) and increased drought tolerance. The transgenic poplar plants exhibited increased wax accumulation and altered wax composition, mainly because of a substantial increase in long-chain (>C30) fatty acids, aldehydes and alkanes. Gene expression analyses revealed that many genes involved in wax biosynthesis were induced in the PeSHN1 overexpression plants. In addition, chromatin immunoprecipitation-PCR assays and dual luciferase assays revealed that at least one of those genes, LACS2, is likely targeted by PeSHN1. Moreover, the PeSHN1 overexpression plants maintained higher photosynthetic activity and accumulated more biomass under drought stress conditions. Taken together, these results suggest that PeSHN1 regulates both WUE and drought tolerance in poplar by modulating wax biosynthesis and that altered PeSHN1 expression could represent a novel approach (altering the wax trait on leaf surfaces to increase WUE) for breeding drought-tolerant plants.

Corrigendum: Populus trichocarpa PtNF-YA9, a Multifunctional Transcription Factor, Regulates Seed Germination, Abiotic Stress, Plant Growth and Development in Arabidopsis.

[This corrects the article DOI: 10.3389/fpls.2018.00954.].

Distinct Carbon and Nitrogen Metabolism of Two Contrasting Poplar Species in Response to Different N Supply Levels.

Poplars have evolved various strategies to optimize acclimation responses to environmental conditions. However, how poplars balance growth and nitrogen deficiency remains to be elucidated. In the present study, changes in root development, carbon and nitrogen physiology, and the transcript abundance of associated genes were investigated in slow-growing Populus simonii (Ps) and fast-growing Populus euramericana (Pe) saplings treated with low, medium, and high nitrogen supply. The slow-growing Ps showed a flourishing system, higher δ15N, accelerated C export, lower N uptake and assimilation, and less sensitive transcriptional regulation in response to low N supply. The slow-growing Ps also had greater resistance to N deficiency due to the transport of photosynthate to the roots and the stimulation of root development, which allows survival. To support its rapid metabolism and growth, compared with the slow-growing Ps, the fast-growing Pe showed greater root development, C/N uptake and assimilation capacity, and more responsive transcriptional regulation with greater N supply. These data suggest that poplars can differentially manage C/N metabolism and photosynthate allocation under different N supply conditions.

Populus trichocarpa PtNF-YA9, A Multifunctional Transcription Factor, Regulates Seed Germination, Abiotic Stress, Plant Growth and Development in Arabidopsis.

NF-YAs play important roles in abiotic stress. However, their characteristics and functions in abiotic stress of poplar, a model woody plant, have not been fully investigated. Here, the biological functions of PtNF-YA9 (Potri.011G101000), an NF-YA gene from Populus trichocarpa, were first fully investigated. PtNF-YA9 is located in the nucleus. The expression of PtNF-YA9 was reduced by mannitol, NaCl, and abscisic acid (ABA). The GUS staining of ProNF-YA9::GUS transgenic lines was also reduced by mannitol treatments. In the PtNF-YA9-overexpressed Arabidopsis (OxPtNA9), OxPtNA9 lines exhibited sensitivity to simulated drought, ABA, and salinity stress during germination stage, and growth arrest emerged at post-germination stage. These phenomena might involve the ABA signaling pathway via the regulation of ABI3, ABI4, and ABI5. At vegetative stages, OxPtNA9 lines decreased in water loss via promoting stomatal closure and displayed high instantaneous water-use efficiency (WUE) of the leaf to exhibit enhanced drought tolerance. Furthermore, OxPtNA9 lines exhibited long primary root in the half-strength Murashige-Skoog agar medium supplemented with NaCl and conferred strong tolerance in the soil under salt stress. Additionally, PtNF-YA9 exhibited dwarf phenotype, short hypocotyl, small leaf area and biomass, delayed flowering, and increased chlorophyll content. Above all, our research proposes a model in which PtNF-YA9 not only plays a key role in reducing plant growth but also can play a primary role in the mechanism of an acclimatization strategy in response to adverse environmental conditions.

Exploration of ABA Responsive miRNAs Reveals a New Hormone Signaling Crosstalk Pathway Regulating Root Growth of Populus euphratica.

Abscisic acid (ABA) plays an important role in the regulation of plant adaptation, seed germination, and root development in plants. However, the mechanism of ABA regulation of root development is still poorly understood, especially through the miRNA-mediated pathway. Here, small RNA (sRNA)-seq and degradome-seq were used to analyze the miRNAs' responsive to ABA in the stems and roots of P. euphratica, a model tree species for abiotic stress-resistance research. In total, 255 unique mature sequences, containing 154 known miRNAs and 101 novel miRNAs were identified, among which 33 miRNAs and 54 miRNAs were responsive to ABA in the roots and stems, respectively. Furthermore, the analysis of these miRNAs and their targets revealed a new hormone signaling crosstalk model of ABA regulation of root growth through miRNA-mediated pathways, such as peu-miR-n68 mediation of the crosstalk between ABA and the brassinosteroid (BR) signaling pathway and peu-miR477b mediation of the crosstalk between ABA and Gibberellic acid (GA) signaling. Taken together, our genome-wide analysis of the miRNAs provides a new insight into the mechanism of ABA regulation of root growth in Populus.

Three stress-responsive NAC transcription factors from Populus euphratica differentially regulate salt and drought tolerance in transgenic plants.

Stress-responsive NAM, Arabidopsis transcription activation factor 1/2 (ATAF1/2) and CUC2 (SNAC) genes are being used to alter stress tolerance in Arabidopsis or grasses through genetic engineering. However, limited reports are available about the functional characteristics of SNAC in trees. In this study, three putative NAC proteins were identified from Populus euphratica. PeNAC034 and PeNAC045 were classified into the ATAF subgroup and PeNAC036 into the ANAC072 subgroup. These three SNAC transcription factors were localized in the nucleus and contained the transcription activation domain in their C-terminal. Under drought and salt stresses, PeNAC036 was strongly induced in the whole plant, but PeNAC034 was significantly suppressed in the roots and stems, and PeNAC045 was inhibited in the roots. PeNAC036 overexpression in Arabidopsis wild-type (WT) (OEPeNAC036) and PeNAC036 complementation in mutant anac072 (anac072/PeNAC036) lines increased tolerance to salt and drought, whereas PeNAC034 overexpression in WT (OEPeNAC034) and PeNAC034 complementation in mutant ataf1 (ataf1/PeNAC034) lines enhanced salt and drought sensitivity. After drought and salt treatments, the expression levels of COR47, RD29B, ERD11, RD22 and DREB2A were upregulated in OEPeNAC036 and anac072/PeNAC036 lines, but were downregulated in OEPeNAC034 and ataf1/PeNAC034 plants. Compared with WT and Vector lines, PeNAC045 overexpression in poplar WT (OEPeNAC045) led to a significant decrease in the net photosynthesis rate, stomatal conductance and transpiration rate under salinity and drought conditions. These results suggest that P. euphratica can adapt to the environment of high salinity and drought, which may be related to the differential expression patterns of SNAC genes.

The role of peu-miR164 and its target PeNAC genes in response to abiotic stress in Populus euphratica.

Plant miR164 family is highly conserved and miR164 members regulate conserved targets belonging to NAC transcription factors. Our previous studies have revealed that peu-miR164a-e and its target gene POPTR_0007s08420 participate in abiotic stress response in Populus euphratica according to deep sequencing and degradome sequencing. In this study, miR164 family comprises six members that generate two mature products (miR164a-e and miR164f) and target seven NAC genes in P. euphratica. Co-expression in Nicotiana benthamiana and 5' RACE confirmed that peu-miR164 directs PeNAC070, PeNAC012 and PeNAC028 mRNAs cleavage. Expression profiles of primary peu-miR164 a/b/c/d/e bear similarity to those of peu-miR164a-e, whereas PeNAC070 and PeNAC081 showed inverse expression patterns with peu-miR164a-e under abiotic stresses. Existence of cis-acting elements in PeNAC070 promoter (ABRE,MBs, Box-W1, GC-motif, and W-box) and in peu-MIR164b promoter (HSE) further confirmed different responses of peu-miR164 and PeNAC070 to abiotic stresses. Histochemical ß-glucuronidase (GUS) staining revealed that GUS activities increased when ProPeNAC070::GUS transgenic Arabidopsis plants were exposed to NaCl, mannitol and abscisic acid (ABA), whereas GUS activity of Propeu-MIR164b::GUS plants decreased under ABA treatment. Subcellular localization and transactivation assays showed that PeNAC070 protein was localized to the nucleus and exhibited transactivation activity at the C-terminal. Overexpression of PeNAC070 in Arabidopsis promoted lateral root development, delayed stem elongation, and increased sensitivity of transgenic plants to drought and salt stresses. This study aids in understanding the adaptability of P. euphratica to extreme drought and salt environment by analysing tissue-specific expression patterns of miR164-regulated and specific promoter-regulated PeNAC genes.

Genome-Wide Analysis of MicroRNA Responses to the Phytohormone Abscisic Acid in Populus euphratica.

MicroRNA (miRNA) is a type of non-coding small RNA with a regulatory function at the posttranscriptional level in plant growth development and in response to abiotic stress. Previous studies have not reported on miRNAs responses to the phytohormone abscisic acid (ABA) at a genome-wide level in Populus euphratica, a model tree for studying abiotic stress responses in woody plants. Here we analyzed the miRNA response to ABA at a genome-wide level in P. euphratica utilizing high-throughput sequencing. To systematically perform a genome-wide analysis of ABA-responsive miRNAs in P. euphratica, nine sRNA libraries derived from three groups (control, treated with ABA for 1 day and treated with ABA for 4 days) were constructed. Each group included three libraries from three individual plantlets as biological replicate. In total, 151 unique mature sequences belonging to 75 conserved miRNA families were identified, and 94 unique sequences were determined to be novel miRNAs, including 56 miRNAs with miRNA(*) sequences. In all, 31 conserved miRNAs and 31 novel miRNAs response to ABA significantly differed among the groups. In addition, 4132 target genes were predicted for the conserved and novel miRNAs. Confirmed by real-time qPCR, expression changes of miRNAs were inversely correlated with the expression profiles of their putative targets. The Populus special or novel miRNA-target interactions were predicted might be involved in some biological process related stress tolerance. Our analysis provides a comprehensive view of how P. euphratica miRNA respond to ABA, and moreover, different temporal dynamics were observed in different ABA-treated libraries.

Genome-wide identification and characterization of the superoxide dismutase gene family in Musa acuminata cv. Tianbaojiao (AAA group).

BACKGROUND: Superoxide dismutase (SOD) is an essential enzyme of the plant antioxidant system that responds to oxidative stresses caused by adverse conditions. Banana is an important staple and economic crop in tropical and subtropical regions. However, its growth and yield are constantly affected by various abiotic stresses. To analyze the roles of distinct SOD genes under various stresses, a detailed characterization and analysis of the SOD gene family in Cavendish banana is indispensable. METHODS: The presence and structure of the SOD family genes were experimentally verified using 5'/3' RACE-PCR, reverse transcription PCR and PCR. Then, their syntenic relationships, conserved motifs and phylogenetic relationships were analyzed using software. Cis-elements present in the promoters were predicted via PlantCARE. And the expression levels under abiotic and hormonal stresses were determined using real-time quantitative polymerase chain reaction. RESULTS: In total, 25 'Tianbaojiao' SOD cDNAs (MaSODs), which encoded six Cu/ZnSODs, four MnSODs and two FeSODs, were cloned. The 12 MaSOD genes were divided into four groups based on their conserved motifs, which corroborated their classifications based on gene-structure patterns and subcellular localizations. Eleven MaSOD promoters were isolated and found to contain many cis-acting elements involved in stress responses. Gene expression analysis showed that 11 out of the 12 MaSODs were expressed in all tested tissues (leaf, pseudostem and root), whereas MaCSD2B was expressed only in leaves and roots. Specific MaSOD members exhibited different expression patterns under abiotic and hormonal treatments. Among the 12 MaSOD genes, MaCSD1D was the only one that responded to all eight treatments, suggesting that this gene plays a predominant role in reactive oxygen species scavenging caused by various stresses in banana. CONCLUSIONS: A genome-wide analysis showed that the 'Tianbaojiao' banana harbored an expanded SOD gene family. Whole genome duplication, segmental duplication and complex transcriptional regulation contributed to the gene expansion and mRNA diversity of the MaSODs. The expression patterns of distinct MaSOD genes showed that they are important responses to different abiotic and hormonal stresses in banana.